WO2005007817A2 - Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides - Google Patents

Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides Download PDF

Info

Publication number
WO2005007817A2
WO2005007817A2 PCT/US2004/021722 US2004021722W WO2005007817A2 WO 2005007817 A2 WO2005007817 A2 WO 2005007817A2 US 2004021722 W US2004021722 W US 2004021722W WO 2005007817 A2 WO2005007817 A2 WO 2005007817A2
Authority
WO
WIPO (PCT)
Prior art keywords
composition
multimeric
synthase
poφhobilinogen
ofthe
Prior art date
Application number
PCT/US2004/021722
Other languages
French (fr)
Other versions
WO2005007817A9 (en
WO2005007817A3 (en
Inventor
Eileen K. Jaffe
Original Assignee
Fox Chase Cancer Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP2006518868A priority Critical patent/JP2007529995A/en
Priority to EP04786044A priority patent/EP1648429A4/en
Priority to AU2004257208A priority patent/AU2004257208B2/en
Priority to CA002531692A priority patent/CA2531692A1/en
Application filed by Fox Chase Cancer Center filed Critical Fox Chase Cancer Center
Publication of WO2005007817A2 publication Critical patent/WO2005007817A2/en
Publication of WO2005007817A3 publication Critical patent/WO2005007817A3/en
Priority to US11/327,762 priority patent/US20060162014A1/en
Priority to IL173016A priority patent/IL173016A/en
Publication of WO2005007817A9 publication Critical patent/WO2005007817A9/en
Priority to US12/106,498 priority patent/US7863029B2/en
Priority to US12/142,435 priority patent/US8153410B2/en
Priority to US12/942,320 priority patent/US20110207816A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/04—Nitro compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • PBGS porphobilinogen synthase
  • LAD 5 -aminolevulinic acid dehydratase
  • PBGS catalyzes the condensation of two 5 -aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen.
  • PBGS was previously understood to be a homooctameric metalloenzyme, which utilizes a variety of divalent and monovalent cations at catalytic and allosteric sites.
  • Mammalian and yeast enzymes typically require Zn(II), some prokaryotic enzymes require either Mg(II) or Zn(II) or both for maximal activity, and plant enzymes seem to require only Mg(II) for enzymatic activity.
  • a small number of organisms have PBGS enzymes that require neither
  • PBGS Additional information regarding PBGS is disclosed in references (3-5) and (7).
  • personal health care products such as wet wipes, diapers, etc. have the ability to not only provide their intended function, but to cure or prevent a disease or a damage caused by contacting bacteria, archaea, and/or eucarya, for example, while not harming the consumer's health.
  • antimicrobial agents have been incorporated into a wide range of consumer products, such as wet wipes, to combat both transient and resident bacteria on skin.
  • Antimicrobial-containing products are currently marketed in many forms such as lotions, deodorant soaps, hard surface cleaners, wet wipes, and surgical disinfectants.
  • antimicrobial agents are harsh or irritating to the skin due to the nature ofthe chemicals utilized to provide the antimicrobial effect.
  • some hard surface cleaners and surgical disinfectants utilize high levels of alcohol and/or surfactants which have been repeatedly shown to dry out and irritate skin tissues.
  • Other wet wipes currently available utilize harsh cationic surfactants without the addition of acids.
  • the surfactant is capable of penetrating and killing many types of bacteria, it is very irritating and harsh to the skin.
  • Many antimicrobial-containing products utilize an organic acid in combination with an anionic or cationic surfactant as antimicrobial agents.
  • biofilms can be a problem for certain surfaces. Biofilms may be found on essentially any environmental surface in which sufficient moisture is present. Their development is most rapid in flowing systems where adequate nutrients are available. Biofilms are composed of populations or communities of microorganisms adhering to environmental surfaces and are complex aggregate of cells and polysaccharide. These microorganisms are usually encased in an extracellular polysaccharide that they synthesize.
  • the biofilm for example can be formed from mixed culture of Pseudomonas aeruginosa, P.fluorescens and Klebsiellapneumoniae.
  • Biofilms may form on solid substrates in contact with moisture, on soft tissue surfaces in living organisms and at liquid air interfaces. Typical locations for biofilm production include rock and other substrate surfaces in marine or freshwater environments. Biofilms are also commonly associated with living organisms, both plant and animal. Tissue surfaces such as teeth and intestinal mucosa which are constantly bathed in a rich aqueous medium rapidly develop a complex aggregation of microorganisms enveloped in an extracellular polysaccharide they themselves produce.
  • Biofilms can be a serious threat to health especially in patients in whom artificial substrates have been introduced. Also, biofilms are a threat to bottoms of ship wherein barnacles can grow and corrode the surface or the internal surface of pipes such as oil pumps or dehumidifiers.
  • an antimicrobial composition having universal applications. It if further desired to provide an agent capable of disturbing an equilibrium of units of multimeric proteins, e.g., an inhibitor capable of inhibiting tetrapyrrole biosynthesis in plants and/or bacteria.
  • the invention provides a composition comprising an agent adapted to affect a multimeric protein by binding to a binding site of said multimeric protein and thereby affecting an equilibrium of units, wherein said multimeric protein comprises an assembly having a plurality of said units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that in said multimeric protein (1) a structure of each of said units determines a structure of said different quaternary isoforms, (2) said units are in the equilibrium and (3) the structure of said different quaternary isoforms influences a
  • affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments, affecting said multimeric protein comprises affecting the function of said multimeric protein.
  • a non-limiting example a function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating.
  • the agent is bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity.
  • each of said units is a member selected from the group consisting of a monomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer.
  • said multimeric protein is a member selected from the group consisting of porphobilinogen synthase and a Class la ribonucleotide reductase.
  • said multimeric protein is porphobilinogen synthase comprising eight porphobilinogen synthase monomers.
  • the active form of the multimeric porphobilinogen synthase has less t than eight monomers.
  • the agent is an inhibitor bound to the quaternary isoform having the lesser activity and wherein the quaternary isoform contains less than eight porphobilinogen synthase monomers.
  • the inhibitor is rosmarinic acid or a derivative thereof.
  • said multimeric protein is the Class la ribonucleotide reductase and the agent inhibits the Class la ribonucleotide reductase through selective binding to the binding site that is unique to the quaternary isoform having the lesser activity.
  • a composition comprising an inhibitor adapted to inhibit formation of an active form of a multimeric porphobilinogen synthase having a first number of monomers by binding to a less active form of the multimeric porphobilinogen synthase having a second number of monomers, wherein the first number of monomers is higher than the second number of monomers.
  • the multimeric porphobilinogen synthase is derived from bacteria, archaea, or eucarya, provided that the octameric porphobilinogen synthase contains an allosteric magnesium binding site. In one variant of this embodiment, the multimeric porphobilinogen synthase contains a catalytic zinc binding site. In certain embodiments, the multimeric porphobilinogen synthase does not contain the allosteric magnesium binding site and the catalytic zinc binding site. In certain embodiments, said less active form is a hexamer. In certain embodiments, wherein said less active form is a dimer.
  • the active form of a multimeric porphobilinogen synthase is an octamer.
  • the inhibitor replaces a metal ion and thereby binds at a metal ion binding site.
  • the metal ion is zinc and/or magnesium.
  • the inhibitor binds at an active site. In certain embodiments, the inhibitor is not a metal cation.
  • the inhibitor is adapted to inhibit formation ofthe active form of the multimeric po ⁇ hobilinogen synthase, said active form is an octomeric po ⁇ hobilinogen synthase by binding to a hug-disabling domain of the less active form of the multimeric porphobilinogen synthase containing less than eight monomers.
  • the inhibitor is adapted to inhibit formation ofthe active form of the multimeric po ⁇ hobilinogen synthase by binding at a site other than an active site and/or metal ion binding site.
  • the inhibitor is adapted to inhibit formation ofthe active form of the multimeric po ⁇ hobilinogen synthase by a mechanism other than removing a metal ion.
  • the composition further comprises a delivery medium, said delivery medium is a member selected from the group consisting of a pharmaceutically- acceptable medium, an orally-acceptable carrier, an antibacterial medium, and a herbicidally- effective medium.
  • the composition is effective to inhibit or prevent formation ofthe active form of the multimeric po ⁇ hobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form ofthe multimeric po ⁇ hobilinogen synthase contains an allosteric magnesium binding site.
  • the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya.
  • the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide.
  • the composition is effective to inhibit or prevent formation of the active form ofthe multimeric po ⁇ hobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form ofthe multimeric po ⁇ hobilinogen synthase does not contain the allosteric magnesium binding site and the catalytic zinc.
  • the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya.
  • the composition is at least one of a drug, a toothpaste, a soap, and a disinfectant.
  • a herbicide resistant plant adapted to be transgenic for a multimeric po ⁇ hobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.
  • the multimeric po ⁇ hobilinogen synthase is derived from a human.
  • the multimeric po ⁇ hobilinogen synthase contains no allosteric magnesium binding site.
  • a composition comprising an inhibitor adapted to bind to a multimeric po ⁇ hobilinogen synthase that does not require zinc for catalytic function.
  • a method of affecting a multimeric protein comprising: providing said multimeric protein comprising an assembly having a plurality of units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of said multimeric protein; providing the composition ofthe invention comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to a binding site on the assembly; and contacting the assembly with the agent, wherein the agent affects the equilibrium by binding to the binding site and thereby affecting said multimeric protein.
  • affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments ofthe method, affecting said multimeric protein comprises affecting a function of said multimeric protein. In certain embodiments of the method, the unit is a member selected from the group consisting of a momomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer. In certain embodiments ofthe method, the agent is adapted to affect a function of said multimeric protein. In certain embodiments of the method, the function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating.
  • the agent is bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity. In certain embodiments of the method, the agent is bound to the quaternary isoform having a greater activity.
  • said multimeric protein is a member selected from the group consisting of po ⁇ hobilinogen synthase and a Class la ribonucleotide reductase. In certain embodiments of the method, said multimeric protein is po ⁇ hobilinogen synthase comprising eight po ⁇ hobilinogen synthase monomers.
  • said multimeric protein is the Class la ribonucleotide reductase and the agent inhibits the Class la ribonucleotide reductase through selective binding to the binding site that is unique to the quaternary isoform having the lesser activity.
  • a method of modulating a physiological activity in a cell, a tissue or an organism comprising: providing the cell, the tissue or the organism, wherein the cell, the tissue or the organism comprise a multimeric protein comprising an assembly having a plurality of units, wherein each of the units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of the multimeric protein; and providing to the cell, the tissue or the organism the composition ofthe invention comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to the binding site on the unit and thereby affecting the formation of a quaternary isoform and thereby modulating the physiological activity.
  • a method of inhibiting a multimeric po ⁇ hobilinogen synthase from forming an active form comprising: applying the composition ofthe invention to the multimeric po ⁇ hobilinogen synthase; associating the composition with the less active form; inhibiting the less active form from assembling into the active form and thereby inhibiting the multimeric po ⁇ hobilinogen synthase from forming the active form.
  • a method for manipulating growth or development of a plant comprising applying the composition ofthe invention which is a herbicide to the plant, wherein the plant is herbicide resistant and is adapted to be transgenic for a multimeric po ⁇ hobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.
  • the multimeric po ⁇ hobilinogen synthase contains no allosteric magnesium binding site.
  • a method of making an antibacterial surface comprising: (1) providing the composition ofthe invention wherein the composition is effective to inhibit or prevent formation ofthe active form ofthe multimeric po ⁇ hobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric po ⁇ hobilinogen synthase contains an allosteric magnesium binding site and the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide; (2) providing a surface-forming matrix; and (3) combining the composition with the surface-forming matrix and thereby making the antibacterial surface.
  • the antibacterial surface is adapted to prevent or inhibit a formation of a biofilm.
  • Fig. IA shows the pH rate profile for wild type human PBGS relative to the F12L variant.
  • Fig. IB shows the chromatographic separation of wild type human (Wt) PBGS and F12L on a mono-Q column.
  • Fig. 1C shows the differential mobility of wild type (Wt) human PBGS and F12L on
  • FIG. 2 A shows schematic representations of a dimer, a tetramer and an octamer of wild type human PBGS.
  • Fig. 2B shows schematic representations of a dimer, a tetramer and a hexamer of human PBGS variant F12L.
  • Fig. 3A shows the chromatographic separation of two peaks of PBGS protein on Q- Sepharose (KC1 gradient (— ), A280 (-)).
  • Fig. 3B shows the differential mobility of two pools of Wt and F12L relative to wild type (Wt) human PBGS and F12L on native gel electrophoresis.
  • Fig. 1 shows schematic representations of a dimer, a tetramer and an octamer of wild type human PBGS.
  • Fig. 2B shows schematic representations of a dimer, a tetramer and a hexamer of human PBGS variant F12L.
  • FIG. 3C shows the pH rate profiles for Pool I (•) and Pool II ( ⁇ ) of Wt and F12L following further purification on Sephacryl S300.
  • Fig. 3D shows a plot of activity versus concentration of ALA for determining the K,,, and V max values for the S300 purified Pool I (circles) and Pool II (squares) at pH 7 (black) and pH 9 (gray).
  • Fig. 4A shows a schematic representation of the crystal structure of E. coli PBGS, including the location ofthe allosteric magnesium.
  • Fig. 4B shows schematic representations of the subunit interfaces of human PBGS octamer (left) relative to the hexamer (right).
  • FIG. 5A shows a schematic representation ofthe equilibrium existing between dimeric, hexameric and octameric PBGS.
  • Fig. 5B shows native gel electrophoresis of pea PBGS, which is isolated in the presence of magnesium, under assay conditions, in the presence of increasing concentrations of EDTA.
  • Fig. 6 shows a classification of PBGS according to the independently segregating criteria of the presence of allosteric magnesium (Mg) (checkered area), the absence of allosteric Mg (white area), the presence of active site zinc (dark grey area), and the absence of active site Zn
  • the resulting matrix (far right) consists of four quadrants, wherein the northwest quadrant (Quadrant NW) represents +Zn/-Mg, the northeast quadrant (Quadrant NE) represents -Zn/-Mg, the southwest quadrant (Quadrant SW) represents +Zn/+Mg, and the southeast quadrant (Quadrant SE) represents -Zn/+Mg.
  • Fig. 7A represents an alignment of active site metal binding residues for the PBGS sequences of Eukaryota and Archaea, which were obtained from GenBank and other web- searchable genomes available as of April 2002.
  • Fig. 7A represents an alignment of active site metal binding residues for the PBGS sequences of Eukaryota and Archaea, which were obtained from GenBank and other web- searchable genomes available as of April 2002.
  • Fig. 7B represents an alignment of active site metal binding residues for the PBGS sequences of Eubacteria, which were obtained from GenBank and other web-searchable genomes available as of April 2002.
  • Fig. 8 is a classification of sources for PBGS including bacteria, archaea, and eucarya, wherein distribution of metal binding properties of PBGS is coded in accordance with Fig. 6.
  • Fig. 9A shows a stereo diagram of one dimer of E. coli PBGS, where the protein subunits are shown as a ribbon diagram. The active site zinc ions are shown as light grey spheres, and the allosteric magnesium ions are shown as black spheres.
  • Fig. 9A shows a stereo diagram of one dimer of E.
  • coli PBGS where the protein subunits are shown as ribbon diagram and colored black and light grey.
  • the active site zinc ions are shown as grey spheres, and the allosteric magnesium ions are shown as black spheres. Active site ligands are not illustrated.
  • Fig. 9B shows a stereo diagram of the structural details of the active site zinc.
  • the cysteine ligands are labeled and the cysteine sulfur atoms are shown as white balls.
  • the water is labeled.
  • the active site ligand, 4,7-DOSA is shown in grey, with oxygen atoms as balls.
  • Fig. 9C shows a stereo diagram of the structural details of the allosteric magesium binding site.
  • the white balls indicate water molecules which form an extended ligation network between the magnesium and oxygen and nitrogen atoms of neighboring residues.
  • the amino acids involved in this network are shown as stick diagrams, with carbons colored light or dark accordinging to the chains of Fig. 9A. Oxygen or nitrogen atoms that are involved in the ligation network are shown in a contrasting shade.
  • the labeled amino acid E231 is the only amino acid in the first coordination sphere ofthe magnesium.
  • Rl 1 derives from the N-terminal arm ofthe neighboring subunit ofthe hugging dimer.
  • FIG. 10 shows a schematic representation of an embodiment ofthe inventive inhibition process, wherein an inhibitor of the invention (represented by circles) binds to one or more domains of the dimeric or hexameric PBGS to inhibit the formation of the octamer, stabilize bound forms and shift equilibrium.
  • Fig. 1 1 is a two-dimensional schematic representation ofthe equilibrium between two isoforms of a protein demonstrating that an agent that is capable of affecting a function ofthe protein has a binding site on one form of a unit but not on another. In each case, the rules for multimerization are to juxapose one thick line with one dashed line.
  • Fig. 1 1 is a two-dimensional schematic representation ofthe equilibrium between two isoforms of a protein demonstrating that an agent that is capable of affecting a function ofthe protein has a binding site on one form of a unit but not on another. In each case, the rules for multimerization are to juxapose one thick line with one dashed line.
  • FIG. 12 is a two-dimensional schematic representation ofthe equilibrium between two isoforms of a protein demonstrating that the equilibrium must go through interconversion of different units.
  • Fig. 13 is a two-dimensional schematic representation of a variety of quaternary isoforms and the equilibrium between units and oligomers. It shows four different configurations of a protein subunit (a multimeric protein).
  • a multimeric protein can be a dimer (shown herein as an oval), a trimer (shown herein as a sphere), and a tetramer (shown herein as a square).
  • a shape of a unit controls a shape of a multimeric protein, e.g., there are units that can form a dimer, a trimer, and a tetramer; also, there are units that cannot oligomerize.
  • This figure shows that a unit has to undergo specific transformations to form oligomers of certain shapes (isoforms).
  • the structure ofthe monomer dictates the oligomeric state and the oligomeric state dictates the functional characteristics.
  • two different conformations ofthe unit must be utilized in order to equilibrate between the multimers.
  • Fig. 14 is a two-dimensional schematic representation ofthe equilibrium between units and oligomers involving an allosteric regulator (agent).
  • the allosteric regulator is shown as a filled gray shape bound to either a unit or to a multimeric protein.
  • the allosteric regulator is capable of perturbing the equilibrium between oligomeric states.
  • Fig. 15 shows the structure S756393 (in spacefill) as it is fit to the model of hexameric P. aeruginosa PBGS.
  • Fig. 16B is a graph featuring specific activity versus concentration of the inhibitor (unfilled circles form a curve wherein no rosmarinic acid was used and filled circles form a curve wherein rosmarinic acid was used at 62.5 mM).
  • This invention provides a novel way to think about how different oligomerization states, such as those involved in signaling and cell cycle control, can up-regulate or down-regulate pathways.
  • the activity of any protein whose allosteric regulation can be defined by an equilibrium among "mo ⁇ heins” can be modulated by agents (e.g., small molecules) that bind to the unique surface features of one or another of these mo ⁇ heins and thus shift the equilibrium of quaternary forms.
  • agents e.g., small molecules
  • tetrapyrrole biosynthesis can be modulated by modulating the equilibrium among the mo ⁇ heins of PBGS.
  • inhibitor molecules can be discovered that will preferentially interact with the unique surface components ofthe PBGS hexamer and displace the distribution of mo ⁇ heins toward the hexameric form (which in the case of plant and some bacterial PBGS is believed to be the inactive form).
  • the invention provides a composition comprising an agent adapted to affect a multimeric protein by binding to a binding site of said multimeric protein and thereby affecting an equilibrium of units, wherein said multimeric protein comprises an assembly having a plurality of said units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that in said multimeric protein (1) a structure of each of said units determines a structure of said different quaternary isoforms, (2) said units are in the equilibrium and (3) the structure of said different quaternary isoforms influences a function ofthe multimeric protein.
  • the composition ofthe invention can be used for inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya in a human or an animal host.
  • the composition ofthe invention can be used in form of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide.
  • the invention provides a guidance to selection of a target organism and influencing it to achieve a desired effect.
  • the unit ofthe multimeric protein can be, for example, a monomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer.
  • affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments, affecting said multimeric protein comprises affecting the function of said multimeric protein.
  • a non-limiting example a function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating.
  • the agent can be bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity, thus inhibiting or activating the multimeric protein.
  • Exemplary multimeric proteins are po ⁇ hobilinogen synthase and a Class la ribonucleotide reductase.
  • the agent is an inhibitor bound to the quaternary isoform having the lesser activity and wherein the quaternary isoform contains less than eight po ⁇ hobilinogen synthase monomers.
  • the agent inhibits the Class la ribonucleotide reductases through selective binding to the binding site that is unique to a less active quaternary isoform.
  • the invention provides a composition
  • a composition comprising an inhibitor adapted to inhibit formation of an active form of a multimeric po ⁇ hobilinogen synthase having a first number of monomers by binding to a less active form ofthe multimeric po ⁇ hobilinogen synthase having a second number of monomers, wherein the first number of monomers is higher than the second number of monomers.
  • PBGS can exist in at least two alternate quaternary structures, octamer and hexamer.
  • the multimeric PBGS having a lesser number of monomers of PBGS is also encompassed by this invention. Previously, only the octameric form was known to exist.
  • the monomer contains an ⁇ 8 ⁇ 8 barrel comprised of the C- terminal 300 amino acids, wherein the center of the ⁇ 8 ⁇ 8 barrel contains the active site.
  • a variable length N-terminal portion of the subunit forms an extended arm structure that is involved in extensive inter-subunit interactions in both oligomeric forms, i.e., octamer and hexamer.
  • a major difference between the two quaternary structures is the conformation ofthe N-terminal arm.
  • the multimeric po ⁇ hobilinogen synthase is derived from bacteria, archaea, or eucarya, provided that the octameric po ⁇ hobilinogen synthase contains an allosteric magnesium binding site.
  • the multimeric po ⁇ hobilinogen synthase contains a catalytic zinc binding site
  • MORPHEINS CONCEPT One dogma of modern biochemistry is that the three dimensional structure of a protein is a direct consequence ofthe amino acid sequence of that protein. Consequently, we are taught that one protein sequence makes one native structure. The discovery of prions challenges the one structure concept, but not if one believes these to be "misfolded". The current invention draws on a new discovery, that of mo ⁇ heins, which are alternate protein quaternary structures that are a physiologically relevant consequence of a conformational change in the monomeric unit.
  • Figs. 11 - 14 The first example of mo ⁇ heins is the po ⁇ hobilinogen synthase (PBGS) system where the quaternary structure equilibrium forms the structural basis for the phenomenon of allosterism.
  • PBGS po ⁇ hobilinogen synthase
  • Figs. 11 - 14 the fundamental structural unit is monomeric and the association of any two units is driven by the placement of a dashed line adjacent to a thick line. This is the rule of assembly.
  • Fig. 11 - 14 the fundamental structural unit is monomeric and the association of any two units is driven by the placement of a dashed line adjacent to a thick line. This is the rule of assembly.
  • FIG. 13 is a two dimensional illustration ofthe concept that the multiplicity of the assembly is directed by the shape ofthe fundamental structural unit (shown as a monomer) and the rule of assembly.
  • the fundamental structural unit shown as a monomer
  • Fig. 13 there are four different shapes for the fundamental unit.-
  • the monomeric pac-man-like shape in the lower left cannot come together with itself in a way that places the dashed line adjacent to the thick line, this monomer cannot oligomerize.
  • the half- oval monomer shape is capable of coming together with itself to form a dimer. Once the dimer is formed, all ofthe dashed lines are adjacent to all ofthe thick lines and oligomerization stops at the dimer.
  • each multimer has different physiologically relevant functional characteristics, such as different Km and Vmax values. For instance, one multimer might be the allosteric "on state” with high enzymatic activity and another multimer might be the allosteric "off state” with low activity. Alternatively, the function ofthe different oligomers might be a result of differences in the molecular surface ofthe oligomer.
  • Allosterism is a general concept wherein the activity of an enzyme is affected by the binding of an allosteric regulator molecule to a binding site on the protein that is not the catalytic site.
  • Most models of allosteric regulation propose that the active and the inactive state are oligomers ofthe same multiplicity, and that these two forms are in equilibrium with each other, and that binding of an allosteric regulator molecule can shift this equilibrium.
  • Fig.11 illustrates this concept using for example the tetramer and trimer shown in Fig 13, but adds a splinter to illustrate the allosteric regulator that can only bind to the tetramer. Binding of the splinter perturbs the quaternary structure equilibrium and draws the system toward the tetrameric form.
  • Fig. 14 illustrates the concept of agents (shown as wedges) that can trap a desired quaternary state ofthe protein and thus act to draw the equilibrium toward that state.
  • these agents which will perturb the quaternary structure equilibrium of mo ⁇ heins, can inhibit or activate the protein.
  • the agent is an inhibitor, which traps the inactive form and therefore prevents formation ofthe active form.
  • the protein is PBGS, the inactive form is a hexamer and the active form is an octamer.
  • the inhibitor is a rosemarinic acid or a derivative thereof.
  • the agent is an activator, which traps the active form ofthe protein.
  • promoter refers to a nucleic acid sequence, usually found upstream (5') to a coding sequence, that controls expression ofthe coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase or other factors necessary for start of transcription at the correct site.
  • mRNA messenger RNA
  • a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences.
  • the promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA.
  • Regeneration refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant).
  • Transformation refers to a process of introducing an exogenous nucleic acid sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is inco ⁇ orated into a chromosome or is capable of autonomous replication.
  • a “transformed cell” is a cell whose DNA has been altered by the introduction of an exogenous nucleic acid molecule into that cell.
  • the term “gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.
  • the phrase "DNA segment heterologous to the promoter region” means that the coding DNA segment does not exist in nature in the same gene with the promoter to which it is now attached.
  • encoding DNA refers to chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA that encodes any ofthe enzymes discussed herein.
  • the term “genome” as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs ofthe present invention introduced into bacterial host cells can therefore be either chromosomally integrated or plasmid-localized.
  • the term “genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components ofthe cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized.
  • herbicide refers to a chemical substance used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues.
  • inhibitor refers to a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the organism.
  • a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the organism.
  • an inhibitor is a chemical substance that inactivates the enzymatic activity of po ⁇ hobilinogen synthase.
  • herbicide is used herein to define an inhibitor when applied to plants, plant cells, plant seeds, or plant tissues.
  • microbe or “microorganism” refer to algae, bacteria, archae, fungi, and protozoa.
  • “Overexpression” refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein.
  • plant refers to any plant or part of a plant at any stage of development. Therein are also included cuttings, cell or tissue cultures and seeds.
  • plant tissue includes, but is not limited to, whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and/or functional units.
  • plastid refers to the class of plant cell organelles that includes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids. These organelles are self-replicating and contain what is commonly referred to as the "chloroplast genome,” a circular DNA molecule that ranges in size from about 120 kb to about 217 kb, depending upon the plant species, and which usually contains an inverted repeat region.
  • solids refers to the nonaqueous component of a tuber (such as in potato) or a fruit (such as in tomato) comprised mostly of starch and other polysaccharides, simple carbohydrates, nonstructural carbohydrated, amino acids, and other organic molecules.
  • tolerance/resistance refers to the ability to continue normal growth or function when exposed to an inhibitor or herbicide.
  • transformation refers to a process for introducing heterologous DNA into a cell, tissue, or plant. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • the "oral composition” is a product, which in the ordinary course of usage, is not intentionally swallowed for pu ⁇ oses of systemic administration of particular therapeutic agents, but is rather retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and/or oral tissues for pu ⁇ oses of oral activity.
  • the oral composition may be a single phase oral composition or may be a combination of two or more oral compositions.
  • orally-acceptable carrier as used herein means a suitable vehicle, which can be used to apply the present compositions to the oral cavity in a safe and effective manner.
  • Such vehicle may include materials such as fluoride ion sources, additional anticalculus agents, buffers, other abrasive materials, peroxide sources, alkali metal bicarbonate salts, thickening materials, humectants, water, surfactants, titanium dioxide, flavor system, sweetening agents, xylitol, coloring agents, and mixtures thereof.
  • fluoride ion sources additional anticalculus agents
  • buffers other abrasive materials
  • peroxide sources alkali metal bicarbonate salts
  • thickening materials e.g., water, surfactants, titanium dioxide, flavor system, sweetening agents, xylitol, coloring agents, and mixtures thereof.
  • mo ⁇ heins are like prions; one protein sequence that can undergo a conformational change which results in an altered quaternary structure (aggregation state).
  • mo ⁇ heins are unlike prions in that the oligomer is of finite mulitplicity and the quaternary structure change is reversible, non-pathologic, and part of a normal physiologic control process. Accordingly, in this the invention, a general mechanism for allosteric regulation using mo ⁇ heins (quaternary structure isoforms) is proposed. In this mechanism, the monomeric structures are different in some aspect of their secondary/tertiary structure, and these differences dictate an assembly into one or the other mo ⁇ hein. This mechanism is illustrated schematically in Figs. 1 1-14.
  • Fig. 11 is a two dimensional representation ofthe equilibrium between two forms of a protein (mo ⁇ heins).
  • a unit (e.g., a monomer) of one form contains four different surfaces, which are a line, a thick line, a dashed line, and a squiggly line.
  • the complementary surfaces that naturally associate are illustrated herein as the thick line with the dashed line. This association defines the rule of engagement between the units.
  • the subunit association potential ofthe square is satisfied (in another words when all thick lines are associated with all dashed lines)
  • the optimal resulting assembly is a tetramer.
  • the oligomeric assembly is dictated by the structure ofthe monomer and the rule of engagement. As shown in Fig.
  • the square structure can associate with a "splinter", which is a schematic representation of an agent (e.g., an allosteric regulator molecule); association of the square monomer and the square tetramer with the splinter affect a function ofthe multimeric protein, for example, in the case of plant and some bacterial PBGS, magnesium provides stability to these forms ofthe protein.
  • the square unit is in equilibrium with another structure, which shares some, but not all of its secondary and tertiary structure and consequently shares only some of the surface characteristics.
  • the alternate unit is illustrated in Fig. 11 as a segment. This monomer contains the surfaces depicted by the thick line and the dashed line; the rule of engagement between these surfaces is the same as for the square unit.
  • the alternate unit assembles into a trimer. It is important that the trimeric structure and its individual components do not contain the binding site for the allosteric regulator molecule (the splinter). Since the splinter stabilizes the square and its oligomer, the presence ofthe splinter wilfpull the equilibrium ofjquaternary structures toward the square and its oligomers.
  • the observation of hexameric PBGS provided the first example of how quaternary structure can serve as a structural basis for allosteric regulation of protein function.
  • Fig. 11 is a general description of the behavior of PBGS.
  • the square is the hugging dimer and the segment is the detached dimer.
  • the structures share some, but not all surface characteristics and the rule of engagement between surfaces is to a first approximation shared between the two alternate structures.
  • the differences in oligomeric structure translate to different functional characteristics. It is reasonable to assume that different quaternary structures of other proteins also translate to different functional characteristics.
  • dimerization of receptors is associated with signal transduction. What has not been appreciated prior to this invention is that the structures ofthe monomer within the dimer structure may not be the same as the structures of the monomer when they are not in the dimer structure.
  • Another non-limiting example of a protein which contains mo ⁇ heins is a Class la ribonucleotide reductase.
  • the recent model put forth for allosteric regulation of Class la ribonucleotide reductase describes an equilibrium between a tetramer and a hexamer (Cooperman and Kashlan, 2003, Adv. In Enzyme Regulation, 43:167-187).
  • the model is schematic only and authors do not have protein structures that define the differences in the putative mo ⁇ heins.
  • ribonucleotide reductase is essential for de novo DNA biosynthesis
  • the Class la enzymes are found in all eucaryotes, and inhibition of de novo DNA biosyntheis is a rational approach to cancer chemotherapy.
  • affecting a function of Class la ribonucleotide reductases e.g., the inhibition
  • the multimetic protein ofthe invention should have at least one characteristic such as a protein concentration dependent specific activity or an ability to separate into different assemblies by e.g., ion-exchange chromatography, native gel eletrophoresis; analitical ultracentrifugation, size -exclusion chromatography (on the basis of size);
  • a protein concentration dependent specific activity or an ability to separate into different assemblies by e.g., ion-exchange chromatography, native gel eletrophoresis; analitical ultracentrifugation, size -exclusion chromatography (on the basis of size);
  • kinetic studies can be conducted to show e.g, K Cincinnati, and V max ; activity as a function of substrate concentration fit to MM equation; mo ⁇ heins will not fit well to a hyperbolic curve but rather a double hyperbolic curve, (see Nature Strucure Biology)
  • a function ofthe multimeric protein is an enzymatic activity and an ability to interact with other molecules such as, for example, an ability
  • the agent is adapted to affect a function of the multimeric protein.
  • Non-limiting examples of the function of the multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating.
  • the agent is associated with the quaternary isoform having a lesser activity.
  • the agent is bound to the quaternary isoform having a greater activity.
  • the octameric form of PBGS binds to substrate in a physiologically relevant concentration range and is active at physiological pH.
  • the octamer is composed of four hugging dimers, where the arms of one subunit hug the barrel of an adjacent subunit with which there are strong barrel-to-barrel interactions.
  • the newly discovered hexameric form of PBGS is an essential component of the regulation of tetrapyrrole biosynthesis in a subset of organisms, including plants and some pathogenic bacteria, but not including humans, animals or fungi.
  • the hexameric form is substantially inactive under physiological conditions.
  • the hexamer cannot bind substrate in the physiologically relevant concentration range because its K,,, value is at least two orders of magnitude larger than the K,,, of the octamer.
  • the hexamer is composed of three detached dimers, where the N-terminal arms do not interact with the adjacent subunit with which there are strong barrel-to-barrel contacts.
  • the transition between the hexameric form and the octameric form involves a significant change in the protein structure. See, e.g., Fig. 5 A.
  • Certain embodiments ofthe invention relate to the inhibition ofthe structural change from a hexamer to an octamer, to inhibit the activation of PBGS and tetrapyrrole biosynthesis in plants and/or bacteria. Since the inhibition mechanism is effective for plants and bacteria, but not animals, the invention provides a novel approach to bacteriostatic, antibiotic and herbicide applications.
  • the invention comprises an inhibitor ofthe hexamer-to- octamer transition for those PBGS that are physiologically regulated by magnesium.
  • the inhibitor can be a known or novel compound.
  • the inhibitor is effective at inhibiting tetrapyrrole biosynthesis in plants and bacterial pathogens at that point in their growth and development where the hexamer-to-octamer transition is physiologically significant. Inhibition of the quaternary structure transition from hexameric PBGS to octameric PBGS is a novel target for the development of antibiotics and herbicides. There is a phylogenetic variation in PBGS proteins where some have an allosteric magnesium and others do not. The PBGS that have the allosteric magnesium are comprised of the archaea, all the bacteria with the exception of the genus Rhodobacter, and all of the photosynthetic eucarya (e.g., green plants) (24).
  • Each E.coli PBGS monomer contains two metal ions, neither of which is phylogenetically conserved.
  • the active site contains a zinc ion that is essential to E.coli PBGS activity but whose three cysteine ligands are not present in many PBGS. This zinc functions in the binding and reactivity ofthe second substrate molecule (33). Details of the zinc site are illustrated in Fig.9B.
  • the first matrix (far left) is divided into two classes: (a) active site zinc on the left (shaded), and (b) no active site zinc on the right (unshaded).
  • the second matrix is divided into two classes: (a) no allosteric magnesium on top (diamonds), and (b) allosteric magnesium on the bottom (squares).
  • Combining the two matrixes provides a matrix (far right) consisting of four quadrants, wherein the northwest quadrant (QNW) represents +Zn/-Mg, the northeast quadrant (QNE) represents -Zn/-Mg, the southwest quadrant (QSW) represents +Zn/+Mg, and the southeast quadrant (QSE) represents -Zn/+Mg.
  • approximately one-half of the currently available sequences encode an active site zinc requirement and one-half do not (i.e., QNW + QSW ⁇ QNE + QSE).
  • PBGS sequences In contrast to the active site metal pattern distribution, more than 90% ofthe PBGS sequences contain the determinants for allosteric magnesium binding (i.e., QSW + QSE » QNW + QNE).
  • the inhibitor will be most effective against a subset of PBGS that contain the allosteric magnesium but do not contain the active site zinc (i.e., PBGS within QSE).
  • PBGS within QSE the photosynthetic eucaryotes and a subset of bacteria, including pathogens such as Pseudomonas aeruginosa.
  • These PBGS proteins elicit the property of protein concentration dependent specific activity, which indicates an interconversion between large active quaternary forms and smaller less active quaternary forms.
  • the inhibitor of the invention is effective to inhibit the formation of octameric PGBS derived from bacteria, archaea, or eucarya, provided that the octameric PGBS contains an allosteric magnesium binding site.
  • Fig. 8 A non-limiting list of sources ofthe octameric PGBS, which can be inhibited by the composition ofthe invention, is shown in Fig. 8, which is a classification of organisms including bacteria, archaea and eucarya. Figs.
  • FIG. 7A and 7B represent an alignment of active site metal binding residues for the PBGS sequences obtained from GenBank and other web-searchable genomes available as of April 2002.
  • the assignment of an organism into one ofthe four quadrants of Fig. 6 is based on the sequence information presented in Fig.7.
  • the presence of the active site zinc binding site is indicated by a cysteine rich cluster (positions 122, 124, and 132 of human PBGS) in association with an arginine residue on the active site lid (position 221 of human PBGS).
  • the inhibitor replaces a metal ion and thereby binds at a metal ion binding site, preferably, the metal ion is zinc or magnesium.
  • the inhibitor binds at an active site.
  • the inhibitor can bind any here, but the binding site must stabilize one quaternary structure. Binding is preferable to a site that is present in one multimer but not the other.
  • Inhibitors ofthe invention can be identified using the following protocol.
  • a model is provided for a hexameric form of a PBGS that contains the allosteric magnesium but does not contain the active site zinc.
  • the initial model can, e.g., be one of pea PBGS.
  • small molecule databases are screened in silico for molecules that will fit into a hug-disabling domain adjacent to the N-terminal portion ofthe subunit.
  • the hug-disabling domain is at least one area of the detached dimer on which binding of the inhibitor inhibits the arms of the dimer from hugging the barrel of that dimer which is necessary to form another dimer to form the active octamer. See Fig. 10, wherein circles represent inhibitors.
  • a likely site of a hug-disabling domain is underneath the joint at which a hugging arm joins the body ofthe subunit (i.e., at the "arm-pit").
  • Theoretically suitable molecules will be empirically tested in vitro by determining their effect on the protein concentration dependent specific activity of pea PBGS, which is available using an artificial gene construct. Those molecules that inhibit the specific activity of the protein in a protein concentration fashion are good inhibitor candidates.
  • the following method will allow identifying inhibitors that will bind anywhere, not necessarily in the hug-disabling domain on PBGS to inhibit octamer formation.
  • a functional assay for specific activity of PBGS will be used first to select potential inhibitors from available molecules that are identified in the computational screen, e.g., substances that are not harmful to humans. After potential inhibitors are selected, they will be further screened for affecting specific activity based on protein concentration.
  • this invention provides a method of affecting a multimeric protein, the method comprising: providing said multimeric protein comprising an assembly having a plurality of units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of said multimeric protein; providing the composition ofthe invention comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to a binding site on the assembly; and contacting the assembly with the agent, wherein the agent affects the equilibrium by binding to the binding site and thereby affecting said multimeric protein.
  • affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments of the method, affecting said multimeric protein comprises affecting a function of said multimeric protein.
  • a method of inhibiting a multimeric po ⁇ hobilinogen synthase from forming an active form comprising: applying the composition ofthe invention to the multimeric po ⁇ hobilinogen synthase; associating the composition with the less active form; inhibiting the less active form from assembling into the active form and thereby inhibiting the multimeric po ⁇ hobilinogen synthase from forming the active form.
  • a non-limiting example of the inhibitor is a rosemarinic acid or derivatives thereof.
  • a preferred application of the inventive composition is for inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya in a human or an animal host.
  • Other applications of the composition of the invention include prevention or inhibition of biofilms on various surfaces including teeth, pipes, tubing ships, or generally any surfaces immersed in water/air mixtures wherein bacteria causing damage can be found.
  • the compositions of the invention can be effective to prevent or inhibit growth of barnacles on a surface of a ship.
  • a composition of the invention can be used to prevent or inhibit damage caused by certain species. Examples of organisms in QSE in the Table so these organisms are primary targets for applying the composition of the invention.
  • composition of the invention can be envisioned such as, for example, a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition and a herbicide.
  • Table 1 Bacteria with PBGS in the SE quadrant
  • the composition ofthe present invention is effective to cure or prevent a disease caused by bacteria, archaea, and/or eucarya.
  • the composition is effective to prevent formation ofthe multimeric PBGS (e.g., octameric PBGS or another active form having a lesser number of nomomers) and thereby inhibit or prevent development or growth of bacteria, archaea, and/or eucarya.
  • the multimeric PBGS contains an allosteric magnesium binding site.
  • the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya.
  • the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide.
  • the composition does not contain the allosteric magnesium binding site and the catalytic zinc.
  • the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya.
  • the composition is at least one of a drug, a toothpaste, a soap, and a disinfectant.
  • Antibiotics, herbicides, and fungicides are often based on the inhibition of an essential pathway that is specific to the bacteria, plant, or fungus and that is not present in humans/animals.
  • the penicillin class of antibiotics is directed against bacterial cell wall biosythesis, and animal cells do not have cell walls
  • the herbicide glyphosate is directed against aromatic amino acid biosynthesis, and humans do not have this pathway, we must eat aromatic amino acids.
  • the phylogenetic variation in metal binding sites among the PBGS of various organisms provides sufficient structural differences for development of an inhibitory agent that will not be inhibitory toward human PBGS.
  • PBGS In the case of PBGS, there are significant differences between organisms in the inherent ability of the PBGS to equilibrate between mo ⁇ hein forms and in the amino acid sequence of the mo ⁇ hein surfaces.
  • the target In the case ofthe more general inhibition of protein function through the selective stabilization of one mo ⁇ hein form, it may be the case that the target is a pathway that is not present in humans or it may be the case that the target simply has sufficient phylogentic variation outside the active site that the surfaces ofthe mo ⁇ heins are very different.
  • the composition comprises a pharmaceutically-acceptable medium in addition to the agent.
  • pharmaceutically-acceptable medium denotes a medium, such as a solvent, that is able to deliver the inhibitor, as well as any other active agents in the composition, to the target organism in a relatively safe and effective manner.
  • the medium itself need not have any pharmaceutical activity.
  • pharmaceutically-acceptable medium includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route.
  • compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the composition ofthe present invention is advantageously administered in the form of i ⁇ jectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared. These preparations also may be emulsified.
  • a typical composition for such pu ⁇ oses comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • aqueous solutions include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well know parameters. Additional formulations are suitable for oral administration.
  • Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • the route is topical, the form may be a cream, ointment, salve or spray.
  • An effective amount ofthe therapeutic agent is determined based on the intended goal.
  • unit dose refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity ofthe therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered depends on the subject to be treated, the state ofthe subject and the protection desired. Precise amounts ofthe therapeutic composition also depend on the judgment ofthe practitioner and are peculiar to each individual.
  • Another application of the inventive composition is a herbicide, wherein the composition additionally comprises a herbicidally-effective medium.
  • “herbicidally-effective medium” denotes a medium, such as a solvent, that is able to deliver the inhibitor, as well as any other active agents in the composition, to the target organism.
  • the medium itself need not have any herbicidal activity.
  • Guidance for applying antibacterial compositions on crops is provided as follows: since all photosynthetic eukaryots fall in the QSE quadrant of Fig 6, they are themselves targets for the inhibitors proposed in this invention. However, the arm-pit inhibitor binding site shown in Fig 10 has significant phylogenetic variation between plants and bacteria. Hence, agents that would act as an antibacterial spray on crops would need to be those that will bind to this site in the bacterial PBGS, but not in the plant PBGS.
  • compositions ofthe present invention include both dilute compositions, which are ready for immediate use, and concentrated compositions, which require to be diluted before use, usually with water.
  • the solid compositions may be in the form of granules, or dusting powders wherein the active ingredient is mixed with a finely divided solid diluent, e.g. kaolin, bentonite, kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth or gypsum or a combination thereof. They may also be in the form of dispersible powders or grains, comprising a wetting agent to facilitate the dispersion ofthe powder or grains in liquid. Solid compositions in the form of a powder may be applied as dusts.
  • Liquid compositions may comprise a solution, suspension, or dispersion ofthe active ingredients in water optionally containing a surface-active agent, or may comprise a solution or dispersion ofthe active ingredient in a water-immiscible organic solvent, which is dispersed as droplets in water.
  • the herbicidal composition is suitable either for tank mixing to produce a dilute composition ready for immediate use or for the formation of a concentrate.
  • the solutions or dispersions may be prepared by dissolving the active ingredients in water or an organic solvent optionally containing wetting or dispersing agent(s) and then, when organic solvents are used, adding the mixture so obtained to water optionally containing wetting or dispersing agent(s).
  • Suitable organic solvents include, for example, ethylene dichloride, isopropyl alcohol, propylene glycol, diacetone alcohol, toluene, kerosene, methylnaphthalene, xylene or trichloroethylene, or a combination thereof.
  • Other additives and adjuvants may also be present in compositions of the present invention. Examples include anti-freeze agents such as ethylene glycol and propylene glycol; dyes; dispersants; rheological agents; anti-foam agents such as silicone based agents; and humectants such as ethylene glycol.
  • HERBICIDE RESISTANT PLANT Further provided is a herbicide resistant plant adapted to be transgenic for a multimeric po ⁇ hobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.
  • the multimeric po ⁇ hobilinogen synthase is derived from a human.
  • the multimeric po ⁇ hobilinogen synthase contains no allosteric magnesium binding site. The following provides a guidance to making the herbicide resistant plant adapted to be transgenic for a multimeric po ⁇ hobilinogen synthase.
  • the expression in a plant of a gene that exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region, which adds polyadenylate nucleotides to the 3' end ofthe RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter.
  • the promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of mRNA.
  • promoter chosen will have the desired tissue and developmental specificity. Therefore, promoter function should be optimized by selecting a promoter with the desired tissue expression capabilities and approximate promoter strength and selecting a transformant that produces the desired PBGS activity. This selection approach from the pool of transformants is routinely employed in expression of heterologous structural genes in plants because there is variation between transformants containing the same heterologous gene due to the site of gene insertion within the plant genome (commonly referred to as "positional effect").
  • the PBGS transgene is to be expressed in the chloroplast in response to light. More specifically, the PBGS transgene is transcribed into mRNA in the nucleus and the mRNA is translated into a precursor polypeptide (Chloroplast Transport Peptide (CTPVPBGS) in the cytoplasm. The precursor polypetide is then transported (imported) into the chloroplast.
  • CTPVPBGS Chroplast Transport Peptide
  • chloroplast light inducible promoters that are active in plant cells have been described in the literature.
  • examples of such promoters include the light-inducible promoter from the small subunit of ribulose-1,5- bisphosphate carboxylase (ssRUBISCO), a very abundant plant polypeptide, the chlorophyll a b binding protein gene promoters and the phytochrome promoter which has been utilized recently in a light-switchable promoter system (Shimizu-Sato et al., 2002).
  • Some of these promoters have been used to create various types of DNA constructs that have been expressed in plants; see, e.g., PCT publication WO 84/02913.
  • promoters that are known to or are found to cause transcription of DNA in plant cells in response to light can be used in the present invention.
  • Such promoters may be obtained from a variety of sources such as plants and plant viruses and include, but are not limited to, the enhanced CaMV35S promoter and promoters isolated from plant genes such as small subunit of ribulose- 1 ,5-biphosphate carboxylase (ssRUBISCO) genes.
  • ssRUBISCO small subunit of ribulose- 1 ,5-biphosphate carboxylase
  • said promoter is leaky in order to provide tetrapyrroles necessary for the non-photosynthetic functions ofthe plant.
  • Plastid-directed Expression of PBGS Activity the PBGS gene is fused to a CTP, in order to target the PBGS protein to the plastid.
  • chloroplast and plastid are intended to include the various forms of plastids including amyloplasts. Many plastid-localized proteins are expressed from nuclear genes as precursors and are targeted to the plastid by a CTP, which is removed during the import steps.
  • chloroplast proteins examples include the small subunit of ribulose-l,5-biphosphate carboxylase (ssRUBISCO, SSU), 5-enolpyruvateshikimate- 3-phosphate synthase (EPSPS), ferredoxin, ferredoxin oxidoreductase, the light-harvesting- complex protein I and protein II, and thioredoxin F.
  • ssRUBISCO small subunit of ribulose-l,5-biphosphate carboxylase
  • EPSPS 5-enolpyruvateshikimate- 3-phosphate synthase
  • ferredoxin ferredoxin
  • ferredoxin oxidoreductase ferredoxin oxidoreductase
  • the light-harvesting- complex protein I and protein II examples include thioredoxin F.
  • the glyphosate-tolerant EPSP synthase plant gene also encodes a polypeptide which contains a CTP, which enables the EPSP synth
  • non-plastid proteins may be targeted to the chloroplast by use of protein fusions with a CTP and that a CTP sequence is sufficient to target a protein to the plastid.
  • a CTP sequence is sufficient to target a protein to the plastid.
  • Those skilled in the art will also recognize that various other chimeric constructs can be made that utilize the functionality of a particular plastid transit peptide to import the PBGS enzyme into the plant cell plastid.
  • the PBGS gene could also be targeted to the plastid by transformation of the gene into the chloroplast genome (Daniell et al., 1998).
  • chloroplast uptake signals such as the CTP are rich in Ser, Thr and small hydrophobic amino acid residues.
  • the RNA produced by a DNA construct ofthe present invention may also contain a 5' non-translated leader sequence.
  • This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase translation of the mRNA.
  • the 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence.
  • the present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequence can be derived from an unrelated promoter or coding sequence. In monocots, an intron is preferably included in the gene construct to facilitate or enhance expression ofthe coding sequence.
  • suitable introns include the HSP70 intron and the rice actin intron, both of which are known in the art.
  • Another suitable intron is the castor bean catalase intron (Suzuki et al., 1994).
  • Polyadenylation Signal The 3' non-translated region ofthe chimeric plant gene contains a polyadenylation signal that functions in plants to cause the addition of polyadenylate nucleotides to the 3' end ofthe
  • RNA examples include (1) the 3' transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean storage protein genes and the small subunit ofthe ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene.
  • Ti Agrobacterium tumor-inducing
  • NOS nopaline synthase
  • ssRUBISCO plant genes like the soybean storage protein genes and the small subunit ofthe ribulose-l,5-bisphosphate carboxylase
  • the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components ofthe desired sequence.
  • the construct Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation ofthe host cell.
  • a recombinant DNA molecule ofthe invention typically includes a selectable marker so that transformed cells can be easily identified and selected from non-transformed cells.
  • nptll neomycin phosphotransferase
  • selectable markers include the bar gene, which confers bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., 1988), which confers glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulphonylurea resistance (European Patent Application 154,204, 1985); and a methotrexate resistant DHFR gene (Thillet et al., 1988). Plants that can be made to express the PBGS transgene include, but are not limited to,
  • Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella et al. (1983), Bevan (1984), Klee et al. (1985) and EPO publication 120,516.
  • Ri root-inducing
  • alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen. DNA may also be inserted into the chloroplast genome (Daniell et al., 1998).
  • a plasmid expression vector suitable for the introduction of a PBGS gene in monocots using microprojectile bombardment is composed ofthe following: a CTP; a light inducible promoter; the PBGS gene; an intron that provides a splice site to facilitate expression ofthe gene, such as the Hsp70 intron (PCT Publication WO93/19189); and a 3' polyadenylation sequence such as the nopaline synthase 3' equence (NOS 3'; Fraley et al., 1983).
  • This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA to be injected into the plant.
  • Plasmid pMON530 is a derivative of pMON505 prepared by transferring the 2.3 kb Stul-Hindlll fragment of pMON316 (Rogers et al., 1987) into pMON526. Plasmid pMON526 is a simple derivative of pMON505 in which the Smal site is removed by digestion with Xmal, treatment with Klenow polymerase and ligation.
  • Plasmid pMON530 retains all the properties of pMON505 and the CaMV35S-NOS expression cassette and now contains a unique cleavage site for Smal between the promoter and polyadenylation signal.
  • Binary vector pMON505 is a derivative of pMON200 (Rogers et al., 1987) in which the i plasmid homology region, LIH, has been replaced with a 3.8 kb Hindlll to Smal segment of the mini RK2 plasmid, pTJS75 (Schmidhauser and Helinski, 1985).
  • Plasmid pMON505 retains all the important features of pMON200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the spectinomycin/streptomycin resistance determinant for selection in E. coli and A. tumefaciens, an intact nopaline synthase gene for facile scoring of transformants and inheritance in progeny, and a pBR322 origin of replication for ease in making large amounts ofthe vector in E.
  • Plasmid pMON505 contains a single T-DNA border derived from the right end ofthe pTiT37 nopaline-type T-DNA. Southern blot analyses have shown that plasmid pMON505 and any DNA that it carries are integrated into the plant genome, that is, the entire plasmid is the T-DNA that is inserted into the plant genome. One end ofthe integrated DNA is located between the right border sequence and the nopaline synthase gene and the other end is between the border sequence and the pBR322 sequences. Another particularly useful Ti plasmid cassette vector is pMON 17227.
  • This vector is described in PCT Publication WO 92/04449 and contains a gene encoding an enzyme conferring glyphosate resistance (denominated CP4), which is an excellent selection marker gene for many plants, including potato and tomato.
  • the gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed from the FMV promoter as described therein.
  • CTP2 Arabidopsis EPSPS chloroplast transit peptide
  • the cells (or protoplasts) are regenerated into whole plants.
  • Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae
  • the PBGS gene is derived from a species in which the PBGS enzyme does not comprise Mg 2+ but comprises Zn 2+ .
  • the species is yeast or human.
  • a mutant PBGS gene is used to generate a transgenic plant.
  • the PBGS gene is introduced into the plant genome by homologous recombination.
  • the wild type human PBGS genomic DNA and full length cDNA which may be used to generate a transgenic plant are shown below:
  • compositions of the invention is suitable as antimicrobial active ingredients in personal care preparations, for example shampoos, bath additives, hair-care products, liquid and solid soaps (based on synthetic surfactants and salts of saturated and/or unsaturated fatty acids), lotions and creams, deodorants, other aqueous or alcoholic solutions, e.g.
  • the invention therefore relates also to a personal care preparation comprising the composition of the invention and optionally cosmetically tolerable carriers or adjuvants as described in U.S. Patent No.6,689,372 to Holzl et al.
  • the composition are to be used in amounts effective to have the antimicrobial effect, i.e. inhibit or prevent microbial activity.
  • the invention provides a method of antimicrobial treatment of skin, mucosa or hair which comprises, contacting the surface ofthe skin, mucosa or hair of a person in need of said antimicrobial treatment with an antimicrobially effective amount ofthe compound ofthe invention.
  • the personal care preparation according to the invention may be formulated as a water- in-oil or oil-in- water emulsion, as an alcoholic or alcohol-containing formulation, as a vesicular dispersion of an ionic or non-ionic amphiphilic lipid, as a gel, a solid stick or as an aerosol formulation.
  • the cosmetically tolerable adjuvant contains, for example, from 5 to 50% of an oily phase, from 5 to 20% of an emulsifier and from 30 to 90% water.
  • the oily phase may contain any oil suitable for cosmetic formulations, e.g.
  • compositions according to the invention may be contained in a wide variety of cosmetic preparations as described in U.S. Patent No. 6,689,372 to Holzl et al. Especially the following preparations, for example, come into consideration: skin-care preparations, e.g. skin- washing and cleansing preparations in the form of tablet-form or liquid soaps, soapless detergents or washing pastes; bath preparations, e.g.
  • lipsticks, lip gloss, lip contour pencils, nail- care preparations such as nail varnish, nail varnish removers, nail hardeners or cuticle removers
  • intimate hygiene preparations e.g. intimate washing lotions or intimate sprays
  • foot-care preparations e.g. foot baths, foot powders, foot creams or foot balsams, special deodorants and antiperspirants or callous-removing preparations
  • light-protective preparations such as sun milks, lotions, creams and oils, sun blocks or tropicals, pre-tanning preparations or after-sun preparations
  • skin-tanning preparations e.g. self-tanning creams
  • depigmenting preparations e.g.
  • preparations for bleaching the skin or skin-lightening preparations e.g. insect-repellents, e.g. insect-repellent oils, lotions, sprays or sticks; deodorants, such as deodorant sprays, pump-action sprays, deodorant gels, sticks or roll-ons; antiperspirants, e.g. antiperspirant sticks, creams or roll-ons; preparations for cleansing and caring for blemished skin, e.g. soapless detergents (solid or liquid), peeling or scrub preparations or peeling masks; hair-removal preparations in chemical form (depilation), e.g.
  • insect-repellents e.g. insect-repellent oils, lotions, sprays or sticks
  • deodorants such as deodorant sprays, pump-action sprays, deodorant gels, sticks or roll-ons
  • antiperspirants e.g. antiperspirant sticks, creams or roll-ons
  • hair-removing powders liquid hair-removing preparations, cream- or paste-form hair-removing preparations, hair-removing preparations in gel form or aerosol foams
  • shaving preparations e.g. shaving soap, foaming shaving creams, non-foaming shaving creams, foams and gels, preshave preparations for dry shaving, aftershaves or after-shave lotions
  • fragrance preparations e.g. fragrances (eau de Cologne, eau de toilette, eau de perfume, perfume de toilette, perfume), perfume oils or cream perfumes
  • dental-care, denture-care and mouth-care preparations e.g.
  • cosmetic hair- treatment preparations e.g. hair-washing preparations in the form of shampoos and conditioners, hair-care preparations, e.g. pretreatment preparations, hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations, e.g. hair-waving preparations for permanent waves (hot wave, mild wave, cold wave), hair- straightening preparations, liquid hair-setting preparations, foams, hairsprays, bleaching preparations; e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams,
  • the oral composition according to the invention may be, for example, in the form of a gel, a paste, a cream or an aqueous preparation (mouthwash).
  • the oral composition according to the invention may also comprise compounds that release fluoride ions which are effective against the formation of caries, for example inorganic fluoride salts, e.g. sodium, potassium, ammonium or calcium fluoride, or organic fluoride salts, e.g. amine fluorides, which are known under the trade name Olafluor.
  • compositions ofthe invention are also suitable for the treatment of textile fibre materials.
  • Such materials are undyed and dyed or printed fibre materials, e.g. of silk, wool, polyamide or polyurethanes, and especially cellulosic fibre materials of all kinds.
  • Such fibre materials are, for example, natural cellulose fibres, such as cotton, linen, jute and hemp, as well as cellulose and regenerated cellulose.
  • Preferred suitable textile fibre materials are made of cotton.
  • the compositions of the invention can also be used in washing and cleaning formulations, e.g. in liquid or powder washing agents or softeners.
  • the compositions of the invention are also suitable for imparting anti-microbial properties to plastics, e.g.
  • compositions of the invention can be used especially also in household and all-pu ⁇ ose cleaners for cleaning and disinfecting hard surfaces.
  • technical products such as paper treatment liquors, printing thickeners of starch or of cellulose derivatives, surface-coatings and paints, can be preserved and provided with antimicrobial properties.
  • the compositions ofthe invention are also suitable for the antimicrobial treatment of wood and for the antimicrobial treatment of leather and the provision of leather with antimicrobial properties.
  • compositions ofthe present invention can be used as an oral composition such as a dentifrice composition in association with an orally-acceptable carrier as described in U.S. Patent No. 6,740,311 to White, Jr., et al.
  • oral composition are toothpastes, tooth powders, prophylaxis pastes, lozenges, gums and the like suitable for humans and animals.
  • the compositions ofthe invention can be used to prepare antimicrobial surfaces.
  • an antibacterial surface comprising: (1) providing the composition of the invention wherein the composition is effective to inhibit or prevent formation ofthe active form ofthe multimeric po ⁇ hobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric po ⁇ hobilinogen synthase contains an allosteric magnesium binding site and the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide; (2) providing a surface-forming matrix; and (3) combining the composition with the surface-forming matrix and thereby making the antibacterial surface.
  • the antibacterial surface is adapted to prevent or inhibit a formation of a biofilm.
  • a surface-forming matrix includes polymers, biodegradable and no-biodegradabe, silicas, ceramics and combinations thereof for mixing, layering or otherwise associating the composition with the matrix.
  • the composition can also be put on the top or a bottom surface ofthe matrix.
  • the multimeric porphobilinogen synthase contains no allosteric magnesium binding site.
  • the invention will be illustrated in more detail with reference to the following Example, but it should be understood that the present invention is not deemed to be limited thereto.
  • the well known quaternary state for PBGS is the octamer, made up of hugging dimers.
  • PBGS neuropeptides
  • some PBGS exist as an equilibrium of quaternary forms as shown by a protein concentration dependence to the specific activity.
  • the protein concentration dependence to the specific activity indicates that a maximally active oligomer can dissociate or reassociate into smaller less active forms. It was previously believed that the smaller, less active forms, were also multiplicities of hugging dimers. Observation and characterization of a stable oligomer of detached dimers was made possible by the fact that PBGS in the QNW do not readily equilibrate between quaternary isoforms.
  • F 12L mutation of human PBGS allowed us to study a stable form of the hexamer and to establish that it was the hexameric property (and not the specific F12L mutation) that dictated the dramatically different functional properties of F12L relative to wild type human PBGS.
  • F 12L is naturally occurring rare allele for human PBGS (3- 5). Described below are studies of human PBGS (both wild type and F12L0 that were heterologously expressed in E. coli and purified by conventional techniques.
  • PROTEIN EXPRESSION The parent human PBGS is the well-characterized N59/C 162A(6). N59 corresponds to the more soluble of two codominant alleles encoding the PBGS protein.
  • C162A is a benign mutation that removes the possibility of a slowly forming aberrant disulfide bond.
  • the artificial gene for N59/C162A is called Wt below.
  • the sense strand primer used for the QuikChange mutagenesis of Wt to the F12L variant was GGCTACCTCCACCCACTGCTTCGGGCC.
  • Several constructs were prepared for the coexpression of Wt and F12L in E. coli. Both the order ofthe genes and the number of promoters were varied, but these variations did not affect the outcome. The construct containing Wt and F 12L under the control of one promoter is described. Plasmid DNA containing Wt (pET3aWt) was digested with BamHl and Ndel to cut out Wt.
  • the pETl 7b vector DNA was linearized by digestion with BamHl and Ndel and ligated with Wt such that the ATG start codon of Wt was 6 basepairs downstream ofthe ribosomal binding site encoded by the vector.
  • the resultant plasmid was transformed into E. coli XL 1 blue.
  • Plasmid DNA (pET17bWt) was prepared and linearized with Spel and Bpul l021. Plasmid DNA containing the gene F12L (pET3aF12L) was digested with Xbal and Bpul 1021 to produce a fragment containing the ribosomal binding site and the gene for F12L.
  • the gene for F12L and the linearized pET17Wt vector were ligated such that the ribosomal binding site of the F12L gene was 35 basepairs downstream ofthe stopcodon of Wt, and the terminator was 52 basepairs downstream ofthe stop codon for the F12L gene.
  • Plasmid pET17bWtF12L was transformed into E.coli XL 1 blue, plasmid DNA was prepared and transformed into E. coli BLR(DE3) for protein expression as previously described (6).
  • PROTEIN PURIFICATION The bulk of the protein purification procedure (cell disruption, ammonium sulfate fractionation, hydrophobic chromatography on Phenyl-Sepharose, anion exchange chromatography, and gel filtration chromatography on Sephacryl S-300) followed the procedures previously described (6) with the exception that a 70 ml Q-Sepharose column was used in place ofthe DEAE agarose column for the anion exchange step.
  • the Q-Sepharose was run at room temperature using 30 mM potassium phosphate, pH 7.0, 10 mM 2-mercaptoethanol, 10 ⁇ M Zn(II), and employed a KC1 gradient as shown in Fig. 3 A.
  • ALA were 10 ⁇ M, 30 ⁇ M, 100 ⁇ M, 300 ⁇ M, 1 mM, 3 mM, and 10 mM and were each done in duplicate. Variations in the concentration of ALA-HCl did not lead to variations in final pH because the stock 0.1 M ALA-HCl was diluted into 0.1 M HCl prior to addition of a constant volume to the assay mixture. All assays were at 37°C for a fixed time using Ehrlich's reagent to determine po ⁇ hobilinogen formed.
  • ANALYTICAL ULTRACENTRIFUGATION Protein samples were dialyzed into 30 mM potassium phosphate, pH 7.5, 0.1 mM DTT, and 10 ⁇ M ZnCl2 just prior to loading into the ultracentrifuge. Loading concentrations were
  • Crystals were formed using the sitting drop method, equal volume of F12L (4.0 mg ml" 1 ) was mixed with the precipitant (0.4 M monoammonium hydrogen phosphate). ALA was added equimolar to the protein subunit concentration and crystals formed in 3-5 days. Diffraction data were collected at 100K on MAR345 image plate detector coupled with RU-200 rotating anode generator equipped with OSMIC optics and operated at 50kV and lOOma. Crystals were cryoprotected before freezing by transferring them at reservoir solutions containing 12%>, 17%, 23%o and 30%> glycerol for 3 min in each solution. A few data sets were collected showing high degree of disorder and lack of any ligand in the active site area.
  • the structure was solved by molecular replacement with the AmoRe program package by using molecule A of human PBGS structure (pdb code 1E51) as an initial model. Refinement was carried out with program CNS .
  • the final model included one dimer of F 12L - molecule A (residues 11-82, 97-124, 140-169, 172-212, 222-330) and B (residues 3-82, 97- 122, 140-169, 172-212, 226-328), one molecule of an intermediate product ofthe catalytic reaction bound in the active site of molecule A, 241 water molecules and two atoms of Zn which appear to have low occupancies.
  • the crystallographic R-factor is 19.9%, R(free) is 28.6% for 2.2A resolution data, and the RMS deviations for bond lengths and bond angles are 0.18A and 2.0°, respectively. All residues belong to allowed conformation regions on the Ramachandran plot.
  • THE PROPERTIES OF HUMAN PBGS VARIANT F12L Human PBGS variant F12L is remarkably different from the wild type protein. Characterization of purified F12L confirmed that the catalytic activity is very low under conditions where wild type human PBGS is most active. However, F 12L exhibits a remarkably altered pH rate profile and shows considerable activity at basic pH values (Fig. 1 A).
  • PBGS exist in different oligomeric states.
  • the molecular weight for the wild type protein and F 12L were found to be 244,000 ⁇ 8,900 and 197,900 ⁇ 6,500 Daltons, respectively.
  • the former is midway between that expected for an octamer and a hexamer, while the latter is midway between that expected for a hexamer and a tetramer.
  • the wild type protein fit best to a three-state model of dimer, hexamer, and octamer at 7.6%, 51%, and 42% respectively, while F 12L fit best to a two-state model of tetramer and hexamer at a ratio of 70%> to 30%>, with octamer absent.
  • F 12L fit best to a two-state model of tetramer and hexamer at a ratio of 70%> to 30%>, with octamer absent.
  • the catalytic core In each subunit the catalytic core resides completely within the barrel and a 20+ amino acid N-terminal arm is involved in extensive subunit interactions.
  • the sequence ofthe catalytic core is phylogenetically conserved, but that of the N-terminal arm is not.
  • the PBGS dimer seen in the octamer (Fig. 2A, top) involves highly conserved barrel-to-barrel contacts and the N-terminal arm of one subunit is hugging the barrel ofthe sister subunit. Hence, this has been referred to as the hugging dimer (2).
  • the side chain of amino acid 12 does not participate in the hugging interaction. Assembly ofthe tetramer, which is by addition of a second hugging dimer rotated 90° around the central axis (Fig.
  • Code 1PV8 reveals a quaternary structure that involves significant rearrangement of the N-terminal arm relative to the ⁇ / ⁇ barrel (Fig. 2B).
  • the dimer retains the aforementioned barrel-to-barrel contacts but the N-terminal arms are detached rather than hugging (Fig. 2B, top).
  • Assembly of the tetramer retains the aforementioned reciprocal interaction between the arm of one subunit and the base of an ⁇ / ⁇ barrel from a neighboring dimer.
  • this association dictates a 120° rotation around the central axis.
  • the new structure of F 12L (2.2 A resolution) contains significant regions of disorder that impede a structural comparison ofthe active site relative to the previously deposited wild type human PBGS structure (PDB code 1E51, 2.83 A resolution).
  • Amino acid 12 does not interact directly with active site residues in either structure. Furthermore, for those amino acids observed in both structures, most are superimposable.
  • the inventor undertook coexpression of F12L and wild type human PBGS.
  • Pool I is comprised of heterohexamers and that Pool II is comprised of heterooctamers.
  • the pH rate profiles are found to be dominated more by the quaternary structure than by the amino acid composition at position 12.
  • the kinetic parameters K m and V max ofthe S300 purified pools were determined at pH
  • Pool I and Pool II are the two pools of PBGS activity eluted from the Q-Sepharose column, as illustrated in Fig. 3A, and following further purification on a Sephacryl S-300 column.
  • K m ⁇ and K m 2 (both mM) are inte ⁇ reted as the K m for the octamer and hexamer, respectively.
  • the reported V max values (in units of ⁇ moles h"l mg" 1 ) reflect the mole fraction of quaternary species under assay conditions, which remains to be determined. Fitted K m values are independent ofthe distribution of quaternary species.
  • the chemistry ofthe PBGS catalyzed reaction requires the formation of at least two Schiff base intermediates (2, 12, 16, 17, 20). Formation of the carbinolamine precursors to these Schiff bases requires that the participating amino groups are uncharged, or that the local pH is above the pK a of the amino groups.
  • One significant structural difference between hexameric and octameric PBGS is the degree of order found in the amino acids that comprise the active site lid.
  • the crystal structure of hexameric PBGS F12L is lacking in density from most ofthe residues that make up the active site lid, thus implying that the hexamer structure destabilizes the closed lid configuration.
  • the PBGS catalyzed reaction cannot proceed until the external pH is above the pK a ofthe amino groups that participate in Schiff base formation.
  • the hexameric structure is proposed to exhibit activity only when the external pH is sufficiently basic to facilitate Schiff base formation.
  • the high Km can also be attributed to destabilization of the active site lid since crystal structures ofthe PBGS octamer show stabilizing interactions between residues on the lid and the substrate molecule that determines the Km value.
  • the current results provide a novel approach to understanding the regulation of PBGS function. As described below, the insight provided from identification of a PBGS hexamer has considerable significance for rethinking the allosteric regulation of PBGS activity in non-human species.
  • Fig.4 A shows the hugging dimer (light ribbon, dark strand) with the allosteric magnesium as black balls, one of which is illustrated with a large white-on-black arrow.
  • the structures of yeast and human PBGS show that the guanidinium group of an arginine resides in the place ofthe allosteric magnesium as illustrated previously (2). This is Arg240 of human PBGS. If one presumes that all PBGS can exist in the hexameric state under appropriate conditions, then the position ofthe allosteric magnesium is pertinent to a hexamer-octamer transition because this metal binding site is present in the octamer (made up of hugging dimers) and absent in the hexamer (made up of detached dimers).
  • Fig. 4B shows the three subunit to subunit interfaces in the PBGS octamer.
  • the black-on-white arrow shows the barrel-to-barrel interface, which is common to both octameric and hexameric PBGS assemblies.
  • the dots-on-black arrow shows the arm-to-base-of- barrel interaction, which is also common to both octameric and hexameric PBGS assemblies.
  • the white-on-black arrow which is analogous to the allosteric magnesium binding site, shows the arm to barrel interaction that is present in the octamer (hugging dimer) and absent in the hexamer (detached dimer). Consistent with the notion that the allosteric magnesium mediates a hexamer-octamer equilibrium is the effect of magnesium on the kinetic parameters of E. coli PBGS. In this case, the addition ofthe allosteric magnesium causes the K m value to decrease from -2 mM to -200 Mm (8), which is remarkably reminiscent ofthe difference between the K m values ofthe hexameric and octameric forms of human PBGS (Table 1).
  • Fig. 5B illustrates that removal of magnesium from pea PBGS disfavors the largest form in favor of a smaller form, where the mobility of the two forms is consistent with that of octamer and hexamer.
  • the hexamer is a putative storage form ofthe PBGS protein because it is less active at physiologic pH and is characterized by a K m value that is well above the physiological concentration of ALA.
  • the octamer is active at physiological pH and has a K m value that is in the proper range of ALA concentrations during active tetrapyrrole biosynthesis.
  • HEXAMERIC HUMAN PBGS REVEALS A NOVEL STRUCTURAL PARADIGM FOR ALLOSTERIC REGULATION OF PROTEIN FUNCTION AND IS THE FIRST EXAMPLE OF A PROTEIN THAT CAN EXIST AS MORPHEINS : Characterization of the human PBGS variant F12L reveals that this point mutation causes a dramatic change in the structure and function of PBGS. This mutation can serve as a precedent for a single amino acid change resulting in significant changes in protein behavior during evolution. The F 12L mutation destabilizes the PBGS octamer and leads to formation of hexamers. The structural transition between octamer and hexamer must proceed through an unprecedented equilibrium containing two different dimer structures.
  • the allosteric magnesium, present in most PBGS has a binding site in the octamer, but not in the hexamer.
  • Native gel data indicate that removal of the allosteric magnesium favors formation of the hexamer over the octamer.
  • the octamer-hexamer transition defines a novel mechanism for metal ion-dependent allosteric regulation of protein function.
  • This invention describes inhibition of protein function through stabilization of the inactive mo ⁇ hein of PBGS and/or any other protein that might be regulated by the interconversion of mo ⁇ heins.
  • the inventor is taking the following approach.
  • the target molecule is one that will selectively bind to the "arm pit" of the hexamer as illustrated by the balls in Fig. 10.
  • the inventor is taking an "in silico" approach of searching molecular libraries for molecules that will bind to the hexameric form of PBGS from the target organisms.
  • the only existing crystal structure on which the inventor bases a model of target hexameric PBGS is that of human PBGS clinical variant F12L, PDB code 1PV8 (Breinig et al.
  • a PBGS octamer for homology model building the alpha, beta-barrel domain of target PBGS.
  • the chosen structure is PDB code 1GZG ( Frere, F., Schubert, W. D., Stauffer, F., Frankenberg, N., Neier, R, Jahn, D., and Heinz, D. W. (2002) JMol Biol 320, 237-247)
  • Reference (20) is a highly ordered, high resolution crystal structures of Pseudomonas aeruginosa PBGS, itself a target for inhibitors that would "trap" the PBGS hexamer.
  • aeruginosa PBGS was built using various capacities of Swiss-PDB Viewer (www.expasy.ch spdbv/mainpage.html) and other programs.
  • the N-terminal arms were removed from the structure file for the 1GZG dimer.
  • the resulting alpha, beta-barrel domains (residues 32-335) were successively overlaid upon the three dimers of hexameric 1PV8 to create a hexameric assembly ofP. aeruginosa PBGS alpha, beta - barrels.
  • aeruginosa PBGS is missing residues 1-9 of subunits A, C, and E as well as residues 1-11 of subunits B, D, and F.
  • the hexameric P. aeruginosa PBGS was the foundation structure for building a model of hexameric pea PBGS using well established published methods as we have done before (Kundrat, L., Martins, J., Stith, L., Dunbrack, R. L., Jr., and Jaffe, E. K. (2003) J Biol Chem 278, 31325-31330) In searching for molecules that will preferentially bind to hexameric PBGS, the following was discovered. Analysis of the hexamer of PBGS shows that the putative
  • inhibitor binding site also referred to as the arm-pit contains elements ofthe three subunits A, B, and E.
  • Subunits A and B comprise the already defined “detached dimer", where we typically depict the bottom subunit (subunit A, Fig. 15) such that the reader is looking directly into the active site in the center ofthe alpha, beta-barrel .
  • Subunit B shares a barrel-to-barrel interface with subunit A.
  • Subunit E shares a mutual interaction with subunit B wherein the N- terminal arm of one subunit is nested into the base ofthe alpha-beta-barrel ofthe other subunit.
  • Fig. 15 shows the docked inhibitor, rosmarinic acid, described below.
  • FIG. 16A open symbols, illustrates the protein concentration dependence of the specific activity of pea PBGS, which shows half maximal activity at 3.5 ⁇ g/ml PBGS.
  • the equilibrium of quaternary isoforms contains about 50% octamer and about 50%) smaller less active isoforms (e.g. hexamers). If an inhibitor acted through preferential binding to these smaller forms, one would expect a more profound inhibition under conditions where the mo ⁇ hein equilibrium contains these smaller forms. In other words, the inhibitor would be expected to shift the protein concentration dependence to a higher protein concentration, which is shown for rosmarinic acid in FIG 16A (see below). Figs.
  • FIG. 16B and 16C show experiments that were done to determine how best to demonstrate this shift in protein concentration dependence.
  • FIG. 16B shows a dose response curve for pea PBGS, which indicates that the IC50 for rosmarinic acid is -63 ⁇ M, when the inhibitor is given 30 minutes to act on the protein prior to the addition of substrate. Not shown is the dependence of the inhibition on the preincubation time, where inhibition by any one concentration of rosmarinic acid increases with increasing preincubation time, showing that rosmarinic acid acts as a slow- binding inhibitor.
  • Fig. 16C shows that once inhibition has taken place, the protein does not recover within a 30 minute assay time.
  • the data obtained in Figs. 16A, 16B, and 16C, were used to choose the appropriate conditions necessary to demonstrate the effect of rosmarinic acid on the protein concentration dependence of pea PBGS, as follows. The closed circles of Fig.
  • FIG. 16A show the protein concentration dependence ofthe specific activity of pea PBGS following a 30 minute treatment with 30 ⁇ M rosmarinic acid, which results in half maximal activity at 13.5 ⁇ g/ml PBGS.
  • the equilibrium of quaternary forms has shifted from 3.5 ⁇ M to 13.5 ⁇ M; under these conditions and it takes 13.5 ⁇ g/ml PBGS to obtain an equilibrium with 50% octamer.
  • Fig. 17 supports this conclusion with native gel electrophoresis data.
  • Lanes 2 shows that pea PBGS will separate into at least two quaternary forms.
  • the mobility on the gel is consistent with pea PBGS existing as an equilibrium of octamer and hexamer (see also FIG 5B).
  • Lanes 1 and 3 show that the equilibrium is shifted to smaller fo ⁇ ns following treatment with rosmarinic acid.
  • Lane 1 shows the effects of a 30 minute incubation of 250 ⁇ M rosmarinic acid on pea PBGS at 142 ⁇ g/ml and lane 3 shows the effect ofthe addition of 10 mM substrate on this quaternary structure equilibrium.
  • the interactions of this biphenyl compound with the "arm pit" ofthe pea PBGS hexamer are predominantly through hydrogen bonds between the protein subunits A, B, and E and the polar moieties of the rosmarinic acid.
  • the protein contains additional hydrogen bonding potential within 4.0 angstroms ofthe rosmarinic acid.
  • a derivative ofthe rosmarinic acid can be made to have an improved binding by adding additional hydrogen bonding potential to the rosmarinic acid molecule. For instance, one could add a hydroxyl group at the 5 position of either phenyl moiety and improve hydrogen bonding to the protein.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Communicable Diseases (AREA)
  • Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Cosmetics (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Compositions for inhibiting multimeric proteins porphobilinogen synthase and Class Ia ribonucleotide reductase, herbicide resistant plants adapted to be transgenic for multimeric porphobilinogen synthase and methods of use.

Description

HEXAMERIC PORPHOBILINOGEN SYNTHASE AS A TARGET FOR THE DEVELOPMENT OF ANTIBIOTICS AND HERBICIDES
CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of provisional Application No.60/485,253, filed July
7, 2003 and provisional Application No. 60/577,312 filed June 4, 2004, which are incorporated herein in their entireties. REFERENCE TO MATERIAL ON COMPACT DISC The Sequence Listing submitted on compact disc is incorporated herein by reference in its entirety. SPECIFICATION BACKGROUND OF THE INVENTION 1. FIELD OF INVENTION This invention relates to the biosynthesis of tetrapyrroles, and more particularly to a mechanism for inhibiting activation of porphobilinogen synthase. 2. DESCRIPTION OF RELATED ART Tetrapyrrole biosynthesis is an essential pathway in animals, plants, and microbes, including bacteria, archae, fungi, protists and viruses. The first common intermediate is 5- aminolevulinic acid (ALA). The enzymatic reactions from ALA to protoporphyrin IX are common to tetrapyrrole biosynthesis in all organisms. The enzyme porphobilinogen synthase (PBGS), also known as 5 -aminolevulinic acid dehydratase (ALAD), is an ancient and highly conserved protein that catalyzes the first common step in the biosynthesis of tetrapyrroles including heme, chlorophyll, vitamin B 12, and cofactor
F430 (1,2). PBGS catalyzes the condensation of two 5 -aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen. PBGS was previously understood to be a homooctameric metalloenzyme, which utilizes a variety of divalent and monovalent cations at catalytic and allosteric sites. Mammalian and yeast enzymes typically require Zn(II), some prokaryotic enzymes require either Mg(II) or Zn(II) or both for maximal activity, and plant enzymes seem to require only Mg(II) for enzymatic activity. A small number of organisms have PBGS enzymes that require neither
Zn(II) nor Mg(II). The difference in the use of metal ions is caused by a variation of residues in the primary structures in at least two metal-binding regions. It has been found that specific activity of PBGS from some sources is dependent on protein concentration. For example, a protein concentration dependence for the specific activity has been seen for B. japonicum, P. aeruginosa, and pea PBGS, but has not been documented for PBGS from E. coli, yeast, or from mammalian sources (22). It is known to inhibit PBGS by removing metals from an active site, e.g., by treating it with efhylenediaminetetraacetic acid (ΕDTA). Additional information regarding PBGS is disclosed in references (3-5) and (7). Today, many consumers are demanding that personal health care products such as wet wipes, diapers, etc. have the ability to not only provide their intended function, but to cure or prevent a disease or a damage caused by contacting bacteria, archaea, and/or eucarya, for example, while not harming the consumer's health. To meet this demand, antimicrobial agents have been incorporated into a wide range of consumer products, such as wet wipes, to combat both transient and resident bacteria on skin. Antimicrobial-containing products are currently marketed in many forms such as lotions, deodorant soaps, hard surface cleaners, wet wipes, and surgical disinfectants. Many products that contain antimicrobial agents, however, are harsh or irritating to the skin due to the nature ofthe chemicals utilized to provide the antimicrobial effect. For example, some hard surface cleaners and surgical disinfectants utilize high levels of alcohol and/or surfactants which have been repeatedly shown to dry out and irritate skin tissues. Other wet wipes currently available utilize harsh cationic surfactants without the addition of acids. Although the surfactant is capable of penetrating and killing many types of bacteria, it is very irritating and harsh to the skin. Many antimicrobial-containing products utilize an organic acid in combination with an anionic or cationic surfactant as antimicrobial agents. Although some organic acids can safely be utilized in products to control microorganisms without the presence of surfactants, most products incorporating only an organic acid have a low efficacy against bacteria unless used at very high concentrations. At very high concentrations, these acids can make the ultimate product uneconomical and can even raise skin irritation concerns. Furter, biofilms can be a problem for certain surfaces. Biofilms may be found on essentially any environmental surface in which sufficient moisture is present. Their development is most rapid in flowing systems where adequate nutrients are available. Biofilms are composed of populations or communities of microorganisms adhering to environmental surfaces and are complex aggregate of cells and polysaccharide. These microorganisms are usually encased in an extracellular polysaccharide that they synthesize. The biofilm, for example can be formed from mixed culture of Pseudomonas aeruginosa, P.fluorescens and Klebsiellapneumoniae. Biofilms may form on solid substrates in contact with moisture, on soft tissue surfaces in living organisms and at liquid air interfaces. Typical locations for biofilm production include rock and other substrate surfaces in marine or freshwater environments. Biofilms are also commonly associated with living organisms, both plant and animal. Tissue surfaces such as teeth and intestinal mucosa which are constantly bathed in a rich aqueous medium rapidly develop a complex aggregation of microorganisms enveloped in an extracellular polysaccharide they themselves produce. The ability of oral bacteria to store iodophilic polysaccharides or glycogen-like molecules inside their cells is associated with dental caries since these storage compounds may extend the time during which lactic acid formation may occur. It is this prolonged exposure to lactic acid which results in decalcification of tooth enamel. People have made use of microbial biofilms, primarily in the area of habitat remediation.
Water treatment plants, waste water treatment plants and septic systems associated with private homes remove pathogens and reduce the amount of organic matter in the water or waste water through interaction with biofilms. On the other hand biofilms can be a serious threat to health especially in patients in whom artificial substrates have been introduced. Also, biofilms are a threat to bottoms of ship wherein barnacles can grow and corrode the surface or the internal surface of pipes such as oil pumps or dehumidifiers. Despite the foregoing developments, it is desired to provide an antimicrobial composition having universal applications. It if further desired to provide an agent capable of disturbing an equilibrium of units of multimeric proteins, e.g., an inhibitor capable of inhibiting tetrapyrrole biosynthesis in plants and/or bacteria. It is further desired to accomplish such inhibition via a mechanism that is not applicable to humans and animals, thereby creating a novel, highly specific, approach to bacteriostatic, antibiotic, or herbicide activity. All references cited herein are incorporated herein by reference in their entireties. BRIEF SUMMARY OF THE INVENTION Accordingly, the invention provides a composition comprising an agent adapted to affect a multimeric protein by binding to a binding site of said multimeric protein and thereby affecting an equilibrium of units, wherein said multimeric protein comprises an assembly having a plurality of said units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that in said multimeric protein (1) a structure of each of said units determines a structure of said different quaternary isoforms, (2) said units are in the equilibrium and (3) the structure of said different quaternary isoforms influences a function of the multimeric protein. In certain embodiments, affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments, affecting said multimeric protein comprises affecting the function of said multimeric protein. A non-limiting example a function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating. In certain embodiments, the agent is bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity. In certain embodiments, each of said units is a member selected from the group consisting of a monomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer. In certain embodiments, said multimeric protein is a member selected from the group consisting of porphobilinogen synthase and a Class la ribonucleotide reductase. In certain embodiments, said multimeric protein is porphobilinogen synthase comprising eight porphobilinogen synthase monomers. In other embodiments, the active form of the multimeric porphobilinogen synthase has less t than eight monomers. In certain embodiments, the agent is an inhibitor bound to the quaternary isoform having the lesser activity and wherein the quaternary isoform contains less than eight porphobilinogen synthase monomers. In certain embodiments, the inhibitor is rosmarinic acid or a derivative thereof. In certain embodiments, said multimeric protein is the Class la ribonucleotide reductase and the agent inhibits the Class la ribonucleotide reductase through selective binding to the binding site that is unique to the quaternary isoform having the lesser activity. Also provided is a composition comprising an inhibitor adapted to inhibit formation of an active form of a multimeric porphobilinogen synthase having a first number of monomers by binding to a less active form of the multimeric porphobilinogen synthase having a second number of monomers, wherein the first number of monomers is higher than the second number of monomers. In certain embodiments, the multimeric porphobilinogen synthase is derived from bacteria, archaea, or eucarya, provided that the octameric porphobilinogen synthase contains an allosteric magnesium binding site. In one variant of this embodiment, the multimeric porphobilinogen synthase contains a catalytic zinc binding site. In certain embodiments, the multimeric porphobilinogen synthase does not contain the allosteric magnesium binding site and the catalytic zinc binding site. In certain embodiments, said less active form is a hexamer. In certain embodiments, wherein said less active form is a dimer. In certain embodiments, the active form of a multimeric porphobilinogen synthase is an octamer. In certain embodiments, the inhibitor replaces a metal ion and thereby binds at a metal ion binding site. In certain embodiments, the metal ion is zinc and/or magnesium. In certain embodiments, the inhibitor binds at an active site. In certain embodiments, the inhibitor is not a metal cation. In certain embodiments, the inhibitor is adapted to inhibit formation ofthe active form of the multimeric poφhobilinogen synthase, said active form is an octomeric poφhobilinogen synthase by binding to a hug-disabling domain of the less active form of the multimeric porphobilinogen synthase containing less than eight monomers. In certain embodiments, the inhibitor is adapted to inhibit formation ofthe active form of the multimeric poφhobilinogen synthase by binding at a site other than an active site and/or metal ion binding site. In certain embodiments, the inhibitor is adapted to inhibit formation ofthe active form of the multimeric poφhobilinogen synthase by a mechanism other than removing a metal ion. In certain embodiments, the composition further comprises a delivery medium, said delivery medium is a member selected from the group consisting of a pharmaceutically- acceptable medium, an orally-acceptable carrier, an antibacterial medium, and a herbicidally- effective medium. Advantageously, the composition is effective to inhibit or prevent formation ofthe active form of the multimeric poφhobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form ofthe multimeric poφhobilinogen synthase contains an allosteric magnesium binding site. In one variant of this embodiment, the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya. In one variant of this embodiment, the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide. In certain embodiments, the composition is effective to inhibit or prevent formation of the active form ofthe multimeric poφhobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form ofthe multimeric poφhobilinogen synthase does not contain the allosteric magnesium binding site and the catalytic zinc. In one variant of this embodiment, the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya. In one variant of this embodiment, the composition is at least one of a drug, a toothpaste, a soap, and a disinfectant. Further provided is a herbicide resistant plant adapted to be transgenic for a multimeric poφhobilinogen synthase that substantially exist in a multimeric form of a hugging dimer. In certain embodiments, the multimeric poφhobilinogen synthase is derived from a human. In certain embodiments, the multimeric poφhobilinogen synthase contains no allosteric magnesium binding site. Further provided is a composition comprising an inhibitor adapted to bind to a multimeric poφhobilinogen synthase that does not require zinc for catalytic function. Also provided is a method of affecting a multimeric protein, the method comprising: providing said multimeric protein comprising an assembly having a plurality of units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of said multimeric protein; providing the composition ofthe invention comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to a binding site on the assembly; and contacting the assembly with the agent, wherein the agent affects the equilibrium by binding to the binding site and thereby affecting said multimeric protein. In certain embodiments of the method, affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments ofthe method, affecting said multimeric protein comprises affecting a function of said multimeric protein. In certain embodiments of the method, the unit is a member selected from the group consisting of a momomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer. In certain embodiments ofthe method, the agent is adapted to affect a function of said multimeric protein. In certain embodiments of the method, the function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating. In certain embodiments ofthe method, the agent is bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity. In certain embodiments of the method, the agent is bound to the quaternary isoform having a greater activity. In certain embodiments of the method, said multimeric protein is a member selected from the group consisting of poφhobilinogen synthase and a Class la ribonucleotide reductase. In certain embodiments of the method, said multimeric protein is poφhobilinogen synthase comprising eight poφhobilinogen synthase monomers. In certain embodiments of the method, said multimeric protein is the Class la ribonucleotide reductase and the agent inhibits the Class la ribonucleotide reductase through selective binding to the binding site that is unique to the quaternary isoform having the lesser activity. Further provided is a method of modulating a physiological activity in a cell, a tissue or an organism, the method comprising: providing the cell, the tissue or the organism, wherein the cell, the tissue or the organism comprise a multimeric protein comprising an assembly having a plurality of units, wherein each of the units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of the multimeric protein; and providing to the cell, the tissue or the organism the composition ofthe invention comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to the binding site on the unit and thereby affecting the formation of a quaternary isoform and thereby modulating the physiological activity. Further provided is a method of inhibiting a multimeric poφhobilinogen synthase from forming an active form, the method comprising: applying the composition ofthe invention to the multimeric poφhobilinogen synthase; associating the composition with the less active form; inhibiting the less active form from assembling into the active form and thereby inhibiting the multimeric poφhobilinogen synthase from forming the active form. Further provided is a method for manipulating growth or development of a plant comprising applying the composition ofthe invention which is a herbicide to the plant, wherein the plant is herbicide resistant and is adapted to be transgenic for a multimeric poφhobilinogen synthase that substantially exist in a multimeric form of a hugging dimer. In one variant ofthe method, the multimeric poφhobilinogen synthase contains no allosteric magnesium binding site. Further provided is a method of making an antibacterial surface, the method comprising: (1) providing the composition ofthe invention wherein the composition is effective to inhibit or prevent formation ofthe active form ofthe multimeric poφhobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric poφhobilinogen synthase contains an allosteric magnesium binding site and the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide; (2) providing a surface-forming matrix; and (3) combining the composition with the surface-forming matrix and thereby making the antibacterial surface. In one variant of the method, the antibacterial surface is adapted to prevent or inhibit a formation of a biofilm. BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein: Fig. IA shows the pH rate profile for wild type human PBGS relative to the F12L variant. Fig. IB shows the chromatographic separation of wild type human (Wt) PBGS and F12L on a mono-Q column. Fig. 1C shows the differential mobility of wild type (Wt) human PBGS and F12L on
12.5% polyacrylamide native gel electrophoresis. Fig. 2 A shows schematic representations of a dimer, a tetramer and an octamer of wild type human PBGS. Fig. 2B shows schematic representations of a dimer, a tetramer and a hexamer of human PBGS variant F12L. Fig. 3A shows the chromatographic separation of two peaks of PBGS protein on Q- Sepharose (KC1 gradient (— ), A280 (-)). Fig. 3B shows the differential mobility of two pools of Wt and F12L relative to wild type (Wt) human PBGS and F12L on native gel electrophoresis. Fig. 3C shows the pH rate profiles for Pool I (•) and Pool II (■) of Wt and F12L following further purification on Sephacryl S300. Fig. 3D shows a plot of activity versus concentration of ALA for determining the K,,, and Vmax values for the S300 purified Pool I (circles) and Pool II (squares) at pH 7 (black) and pH 9 (gray). Fig. 4A shows a schematic representation of the crystal structure of E. coli PBGS, including the location ofthe allosteric magnesium. Fig. 4B shows schematic representations of the subunit interfaces of human PBGS octamer (left) relative to the hexamer (right). Fig. 5A shows a schematic representation ofthe equilibrium existing between dimeric, hexameric and octameric PBGS. Fig. 5B shows native gel electrophoresis of pea PBGS, which is isolated in the presence of magnesium, under assay conditions, in the presence of increasing concentrations of EDTA. Fig. 6 shows a classification of PBGS according to the independently segregating criteria of the presence of allosteric magnesium (Mg) (checkered area), the absence of allosteric Mg (white area), the presence of active site zinc (dark grey area), and the absence of active site Zn
(white area). The resulting matrix (far right) consists of four quadrants, wherein the northwest quadrant (Quadrant NW) represents +Zn/-Mg, the northeast quadrant (Quadrant NE) represents -Zn/-Mg, the southwest quadrant (Quadrant SW) represents +Zn/+Mg, and the southeast quadrant (Quadrant SE) represents -Zn/+Mg. Fig. 7A represents an alignment of active site metal binding residues for the PBGS sequences of Eukaryota and Archaea, which were obtained from GenBank and other web- searchable genomes available as of April 2002. Fig. 7B represents an alignment of active site metal binding residues for the PBGS sequences of Eubacteria, which were obtained from GenBank and other web-searchable genomes available as of April 2002. Fig. 8 is a classification of sources for PBGS including bacteria, archaea, and eucarya, wherein distribution of metal binding properties of PBGS is coded in accordance with Fig. 6. Fig. 9A shows a stereo diagram of one dimer of E. coli PBGS, where the protein subunits are shown as a ribbon diagram. The active site zinc ions are shown as light grey spheres, and the allosteric magnesium ions are shown as black spheres. Fig. 9A shows a stereo diagram of one dimer of E. coli PBGS, where the protein subunits are shown as ribbon diagram and colored black and light grey. The active site zinc ions are shown as grey spheres, and the allosteric magnesium ions are shown as black spheres. Active site ligands are not illustrated. Fig. 9B shows a stereo diagram of the structural details of the active site zinc. The cysteine ligands are labeled and the cysteine sulfur atoms are shown as white balls. The water is labeled. The active site ligand, 4,7-DOSA is shown in grey, with oxygen atoms as balls. Fig. 9C shows a stereo diagram of the structural details of the allosteric magesium binding site. The white balls indicate water molecules which form an extended ligation network between the magnesium and oxygen and nitrogen atoms of neighboring residues. The amino acids involved in this network are shown as stick diagrams, with carbons colored light or dark acording to the chains of Fig. 9A. Oxygen or nitrogen atoms that are involved in the ligation network are shown in a contrasting shade. The labeled amino acid E231 is the only amino acid in the first coordination sphere ofthe magnesium. Rl 1 derives from the N-terminal arm ofthe neighboring subunit ofthe hugging dimer. Fig. 10 shows a schematic representation of an embodiment ofthe inventive inhibition process, wherein an inhibitor of the invention (represented by circles) binds to one or more domains of the dimeric or hexameric PBGS to inhibit the formation of the octamer, stabilize bound forms and shift equilibrium. Fig. 1 1 is a two-dimensional schematic representation ofthe equilibrium between two isoforms of a protein demonstrating that an agent that is capable of affecting a function ofthe protein has a binding site on one form of a unit but not on another. In each case, the rules for multimerization are to juxapose one thick line with one dashed line. Fig. 12 is a two-dimensional schematic representation ofthe equilibrium between two isoforms of a protein demonstrating that the equilibrium must go through interconversion of different units. Fig. 13 is a two-dimensional schematic representation of a variety of quaternary isoforms and the equilibrium between units and oligomers. It shows four different configurations of a protein subunit (a multimeric protein). A multimeric protein can be a dimer (shown herein as an oval), a trimer (shown herein as a sphere), and a tetramer (shown herein as a square). A shape of a unit controls a shape of a multimeric protein, e.g., there are units that can form a dimer, a trimer, and a tetramer; also, there are units that cannot oligomerize. This figure shows that a unit has to undergo specific transformations to form oligomers of certain shapes (isoforms). In each case, the structure ofthe monomer dictates the oligomeric state and the oligomeric state dictates the functional characteristics. In each case, two different conformations ofthe unit must be utilized in order to equilibrate between the multimers. Fig. 14 is a two-dimensional schematic representation ofthe equilibrium between units and oligomers involving an allosteric regulator (agent). The allosteric regulator is shown as a filled gray shape bound to either a unit or to a multimeric protein. The allosteric regulator is capable of perturbing the equilibrium between oligomeric states. Fig. 15 shows the structure S756393 (in spacefill) as it is fit to the model of hexameric P. aeruginosa PBGS. Fig. 16 A is a graph showing A555 values measured at various assay times (the unfilled circles form a curve wherein no inhibitor was used (Kd = 3.5 μg/ml) and the filled circles form a curve wherein rosmarinic acid was used (Kd = 13.5 μg /ml).
Fig. 16B is a graph featuring specific activity versus concentration of the inhibitor (unfilled circles form a curve wherein no rosmarinic acid was used and filled circles form a curve wherein rosmarinic acid was used at 62.5 mM). Fig. 16C shows that inhibition is associated with stabilization of a smaller oligomer form of pea PBGS (Pea PBGS at 10 mg/ml, 30 min preincubation with rosmarinic acid. IC50 = 62.5 mM). DETAILED DESCRIPTION OF THE INVENTION The present invention was prompted by the inventor's studies relating to the basic science of tetrapyrrole biosynthesis. This invention provides a novel way to think about how different oligomerization states, such as those involved in signaling and cell cycle control, can up-regulate or down-regulate pathways. In accordance with the invention, the activity of any protein whose allosteric regulation can be defined by an equilibrium among "moφheins" can be modulated by agents (e.g., small molecules) that bind to the unique surface features of one or another of these moφheins and thus shift the equilibrium of quaternary forms. According to certain embodiments of the invention, tetrapyrrole biosynthesis can be modulated by modulating the equilibrium among the moφheins of PBGS. According to certain embodiments of the invention, inhibitor molecules can be discovered that will preferentially interact with the unique surface components ofthe PBGS hexamer and displace the distribution of moφheins toward the hexameric form (which in the case of plant and some bacterial PBGS is believed to be the inactive form). Advantegeously, the invention provides a composition comprising an agent adapted to affect a multimeric protein by binding to a binding site of said multimeric protein and thereby affecting an equilibrium of units, wherein said multimeric protein comprises an assembly having a plurality of said units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that in said multimeric protein (1) a structure of each of said units determines a structure of said different quaternary isoforms, (2) said units are in the equilibrium and (3) the structure of said different quaternary isoforms influences a function ofthe multimeric protein. The composition ofthe invention can be used for inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya in a human or an animal host. For example, the composition ofthe invention can be used in form of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide. The invention provides a guidance to selection of a target organism and influencing it to achieve a desired effect. The unit ofthe multimeric protein can be, for example, a monomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer. In certain embodiments, affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments, affecting said multimeric protein comprises affecting the function of said multimeric protein. A non-limiting example a function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating. As described further below, depending on the application, the agent can be bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity, thus inhibiting or activating the multimeric protein. Exemplary multimeric proteins are poφhobilinogen synthase and a Class la ribonucleotide reductase. Thus, in certain embodiments, the agent is an inhibitor bound to the quaternary isoform having the lesser activity and wherein the quaternary isoform contains less than eight poφhobilinogen synthase monomers. Similarly, when the multimeric protein is the Class la ribonucleotide reductase, the agent inhibits the Class la ribonucleotide reductases through selective binding to the binding site that is unique to a less active quaternary isoform. The invention will now be described using PBGS as an example of a multimeric protein. Thus, the invention provides a composition comprising an inhibitor adapted to inhibit formation of an active form of a multimeric poφhobilinogen synthase having a first number of monomers by binding to a less active form ofthe multimeric poφhobilinogen synthase having a second number of monomers, wherein the first number of monomers is higher than the second number of monomers. The inventor has discovered that PBGS can exist in at least two alternate quaternary structures, octamer and hexamer. The multimeric PBGS having a lesser number of monomers of PBGS is also encompassed by this invention. Previously, only the octameric form was known to exist. In both forms, the monomer contains an α8β8 barrel comprised of the C- terminal 300 amino acids, wherein the center of the α8β8 barrel contains the active site. A variable length N-terminal portion of the subunit forms an extended arm structure that is involved in extensive inter-subunit interactions in both oligomeric forms, i.e., octamer and hexamer. A major difference between the two quaternary structures is the conformation ofthe N-terminal arm. In certain embodiments, the multimeric poφhobilinogen synthase is derived from bacteria, archaea, or eucarya, provided that the octameric poφhobilinogen synthase contains an allosteric magnesium binding site. In one variant of this embodiment, the multimeric poφhobilinogen synthase contains a catalytic zinc binding site
The following concepts and definitions are provided in order to aid those skilled in the art in understanding the detailed description ofthe present invention. MORPHEINS CONCEPT: One dogma of modern biochemistry is that the three dimensional structure of a protein is a direct consequence ofthe amino acid sequence of that protein. Consequently, we are taught that one protein sequence makes one native structure. The discovery of prions challenges the one structure concept, but not if one believes these to be "misfolded". The current invention draws on a new discovery, that of moφheins, which are alternate protein quaternary structures that are a physiologically relevant consequence of a conformational change in the monomeric unit. In the case of moφheins, the alternate native states are close in energy to each other, but each state dictates a different finite quaternary multiplicity, as illustrated in schematically in Figs. 11 - 14. The first example of moφheins is the poφhobilinogen synthase (PBGS) system where the quaternary structure equilibrium forms the structural basis for the phenomenon of allosterism. In Figs. 11 - 14, the fundamental structural unit is monomeric and the association of any two units is driven by the placement of a dashed line adjacent to a thick line. This is the rule of assembly. Fig. 13 is a two dimensional illustration ofthe concept that the multiplicity of the assembly is directed by the shape ofthe fundamental structural unit (shown as a monomer) and the rule of assembly. In Fig. 13 there are four different shapes for the fundamental unit.- The monomeric pac-man-like shape in the lower left cannot come together with itself in a way that places the dashed line adjacent to the thick line, this monomer cannot oligomerize. The half- oval monomer shape is capable of coming together with itself to form a dimer. Once the dimer is formed, all ofthe dashed lines are adjacent to all ofthe thick lines and oligomerization stops at the dimer. The pie- wedge shape can come together with itself in the same fashion, but one needs three units in order to have all the dashed lines adjacent to all the thick lines. This the pie- wedge monomer is fated to multimerize into a trimer. Finally, following the same logic, the square monomer form is fated to form a tetrameric assembly. Working within the moφhein concept, each multimer has different physiologically relevant functional characteristics, such as different Km and Vmax values. For instance, one multimer might be the allosteric "on state" with high enzymatic activity and another multimer might be the allosteric "off state" with low activity. Alternatively, the function ofthe different oligomers might be a result of differences in the molecular surface ofthe oligomer. For instance, the rounded surface ofthe trimer in Figs. 11 - 14 would interact with different receptors or binding partners than the oval surface of the dimer or the pointed surface ofthe tetramer. These molecular surface differences could dictate the cellular localization for the complex. Allosterism is a general concept wherein the activity of an enzyme is affected by the binding of an allosteric regulator molecule to a binding site on the protein that is not the catalytic site. Most models of allosteric regulation propose that the active and the inactive state are oligomers ofthe same multiplicity, and that these two forms are in equilibrium with each other, and that binding of an allosteric regulator molecule can shift this equilibrium. In general, there is insufficient structural information (three dimensional X-ray crystal structures for instance) in order to allow an understanding of where the allosteric regulator binds, how the two forms differ from each other, why one form is more active than the other. We recently discovered two different quaternary forms ofthe enzyme poφhobilinogen synthase (PBGS) wherein one can examine these forms and deduce a rational explanation for the allosteric regulation ofthe PBGS of some organisms by magnesium. This discovery is the first concrete example of moφheins and is described in detail in the body of this application. However, the observation of alternate quaternary forms of PBGS leads to a general description of the concept of moφheins as a regulatory mechanism for protein function and the description of agents that can trap one or another ofthe moφhein forms such as to direct protein function. Fig.11 illustrates this concept using for example the tetramer and trimer shown in Fig 13, but adds a splinter to illustrate the allosteric regulator that can only bind to the tetramer. Binding of the splinter perturbs the quaternary structure equilibrium and draws the system toward the tetrameric form. Fig. 14 illustrates the concept of agents (shown as wedges) that can trap a desired quaternary state ofthe protein and thus act to draw the equilibrium toward that state. Thus, these agents, which will perturb the quaternary structure equilibrium of moφheins, can inhibit or activate the protein. In one embodiment ofthe invention, the agent is an inhibitor, which traps the inactive form and therefore prevents formation ofthe active form. In one embodiment, the protein is PBGS, the inactive form is a hexamer and the active form is an octamer. A non-limiting example ofthe inhibitor is a rosemarinic acid or a derivative thereof. In another embodimentof the invention, the agent is an activator, which traps the active form ofthe protein. The term "promoter" or "promoter region" refers to a nucleic acid sequence, usually found upstream (5') to a coding sequence, that controls expression ofthe coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase or other factors necessary for start of transcription at the correct site. As contemplated herein, a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences. The promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA. "Regeneration" refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant). "Transformation" refers to a process of introducing an exogenous nucleic acid sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is incoφorated into a chromosome or is capable of autonomous replication. A "transformed cell" is a cell whose DNA has been altered by the introduction of an exogenous nucleic acid molecule into that cell. The term "gene" refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression. The phrase "DNA segment heterologous to the promoter region" means that the coding DNA segment does not exist in nature in the same gene with the promoter to which it is now attached. The term "encoding DNA" refers to chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA that encodes any ofthe enzymes discussed herein. The term "genome" as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs ofthe present invention introduced into bacterial host cells can therefore be either chromosomally integrated or plasmid-localized. The term "genome" as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components ofthe cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized. The term "herbicide" refers to a chemical substance used to kill or suppress the growth of plants, plant cells, plant seeds, or plant tissues. The term "inhibitor" refers to a chemical substance that inactivates the enzymatic activity of a protein such as a biosynthetic enzyme, receptor, signal transduction protein, structural gene product, or transport protein that is essential to the growth or survival of the organism. In the context of the instant invention, an inhibitor is a chemical substance that inactivates the enzymatic activity of poφhobilinogen synthase. The term "herbicide" is used herein to define an inhibitor when applied to plants, plant cells, plant seeds, or plant tissues. The terms "microbe" or "microorganism" refer to algae, bacteria, archae, fungi, and protozoa. "Overexpression" refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein. The term "plant" refers to any plant or part of a plant at any stage of development. Therein are also included cuttings, cell or tissue cultures and seeds. As used in conjunction with the present invention, the term "plant tissue" includes, but is not limited to, whole plants, plant cells, plant organs, plant seeds, protoplasts, callus, cell cultures, and any groups of plant cells organized into structural and/or functional units. The term "plastid" refers to the class of plant cell organelles that includes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids. These organelles are self-replicating and contain what is commonly referred to as the "chloroplast genome," a circular DNA molecule that ranges in size from about 120 kb to about 217 kb, depending upon the plant species, and which usually contains an inverted repeat region. The term "solids" refers to the nonaqueous component of a tuber (such as in potato) or a fruit (such as in tomato) comprised mostly of starch and other polysaccharides, simple carbohydrates, nonstructural carbohydrated, amino acids, and other organic molecules. The term "tolerance/resistance" refers to the ability to continue normal growth or function when exposed to an inhibitor or herbicide. The term "transformation" refers to a process for introducing heterologous DNA into a cell, tissue, or plant. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. The "oral composition" is a product, which in the ordinary course of usage, is not intentionally swallowed for puφoses of systemic administration of particular therapeutic agents, but is rather retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and/or oral tissues for puφoses of oral activity. The oral composition may be a single phase oral composition or may be a combination of two or more oral compositions. The term "orally-acceptable carrier" as used herein means a suitable vehicle, which can be used to apply the present compositions to the oral cavity in a safe and effective manner. Such vehicle may include materials such as fluoride ion sources, additional anticalculus agents, buffers, other abrasive materials, peroxide sources, alkali metal bicarbonate salts, thickening materials, humectants, water, surfactants, titanium dioxide, flavor system, sweetening agents, xylitol, coloring agents, and mixtures thereof. The term "moφheins" is used in this disclosure by the inventor to describe different quaternary isoforms of a protein with different functional characteristics (e.g., different catalytic properties). Additional terms for these quaternary structure isoforms include "quatreins",
"isoquatomers" and "selkeins". The latter term is chosen after the mythic selkie that can change form and function in response to specific stimuli. In the case of plant and some bacterial PBGS, the stimulus for transition between moφheins is the allosteric regulator, i.e., an agent (e.g., magnesium). Moφheins of a given protein have partial differences in secondary and tertiary structure, and these differences dictate a difference in quaternary structure. In certain aspects, moφheins are like prions; one protein sequence that can undergo a conformational change which results in an altered quaternary structure (aggregation state). However, in other aspects, moφheins are unlike prions in that the oligomer is of finite mulitplicity and the quaternary structure change is reversible, non-pathologic, and part of a normal physiologic control process. Accordingly, in this the invention, a general mechanism for allosteric regulation using moφheins (quaternary structure isoforms) is proposed. In this mechanism, the monomeric structures are different in some aspect of their secondary/tertiary structure, and these differences dictate an assembly into one or the other moφhein. This mechanism is illustrated schematically in Figs. 1 1-14. Fig. 11 is a two dimensional representation ofthe equilibrium between two forms of a protein (moφheins). A unit (e.g., a monomer) of one form (shown herein as a square) contains four different surfaces, which are a line, a thick line, a dashed line, and a squiggly line. The complementary surfaces that naturally associate are illustrated herein as the thick line with the dashed line. This association defines the rule of engagement between the units. When the subunit association potential ofthe square is satisfied (in another words when all thick lines are associated with all dashed lines), the optimal resulting assembly is a tetramer. Thus, the oligomeric assembly is dictated by the structure ofthe monomer and the rule of engagement. As shown in Fig. 11 , the square structure can associate with a "splinter", which is a schematic representation of an agent (e.g., an allosteric regulator molecule); association of the square monomer and the square tetramer with the splinter affect a function ofthe multimeric protein, for example, in the case of plant and some bacterial PBGS, magnesium provides stability to these forms ofthe protein. The square unit is in equilibrium with another structure, which shares some, but not all of its secondary and tertiary structure and consequently shares only some of the surface characteristics. The alternate unit is illustrated in Fig. 11 as a segment. This monomer contains the surfaces depicted by the thick line and the dashed line; the rule of engagement between these surfaces is the same as for the square unit. Consequently, following this rule of engagement, the alternate unit assembles into a trimer. It is important that the trimeric structure and its individual components do not contain the binding site for the allosteric regulator molecule (the splinter). Since the splinter stabilizes the square and its oligomer, the presence ofthe splinter wilfpull the equilibrium ofjquaternary structures toward the square and its oligomers. The observation of hexameric PBGS provided the first example of how quaternary structure can serve as a structural basis for allosteric regulation of protein function. In PBGS from photosynthetic organisms and some bacteria, a protein concentration dependence of the specific activity provides evidence for an equilibration between a fully active (presumably octameric) form and an inactive (presumably hexameric) form (See Fig. 5A). Fig. 11 is a general description of the behavior of PBGS. In the case of PBGS, the square is the hugging dimer and the segment is the detached dimer. In each case, the structures share some, but not all surface characteristics and the rule of engagement between surfaces is to a first approximation shared between the two alternate structures. In the case of PBGS, the differences in oligomeric structure translate to different functional characteristics. It is reasonable to assume that different quaternary structures of other proteins also translate to different functional characteristics. It is well known that dimerization of receptors is associated with signal transduction. What has not been appreciated prior to this invention is that the structures ofthe monomer within the dimer structure may not be the same as the structures of the monomer when they are not in the dimer structure. Another non-limiting example of a protein which contains moφheins is a Class la ribonucleotide reductase. The recent model put forth for allosteric regulation of Class la ribonucleotide reductase describes an equilibrium between a tetramer and a hexamer (Cooperman and Kashlan, 2003, Adv. In Enzyme Regulation, 43:167-187). In this case, the model is schematic only and authors do not have protein structures that define the differences in the putative moφheins. However, ribonucleotide reductase is essential for de novo DNA biosynthesis, the Class la enzymes are found in all eucaryotes, and inhibition of de novo DNA biosyntheis is a rational approach to cancer chemotherapy. Thus, affecting a function of Class la ribonucleotide reductases (e.g., the inhibition) can be achieved through selective binding of an effector to a surface that is unique to the less active moφhein. The multimetic protein ofthe invention should have at least one characteristic such as a protein concentration dependent specific activity or an ability to separate into different assemblies by e.g., ion-exchange chromatography, native gel eletrophoresis; analitical ultracentrifugation, size -exclusion chromatography (on the basis of size); To demonstrate the equilibrium, kinetic studies can be conducted to show e.g, K„, and Vmax; activity as a function of substrate concentration fit to MM equation; moφheins will not fit well to a hyperbolic curve but rather a double hyperbolic curve, (see Nature Strucure Biology) Non-limiting example of a function ofthe multimeric protein is an enzymatic activity and an ability to interact with other molecules such as, for example, an ability to bind a different _. protein. The function can be inhibited or enhanced. Monitoring of changes in the function can be conducted by, for example, monitoring kinetic parameters K,n and Vmax as a skilled artesian would appreciate. In certain embodiments of the invention, inhibition of a protein function through stabilization of a less active moφhein. In certain embodiments, the agent is adapted to affect a function of the multimeric protein. Non-limiting examples of the function of the multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating. In certain embodiments, the agent is associated with the quaternary isoform having a lesser activity. In certain embodiments, the agent is bound to the quaternary isoform having a greater activity. A non-limiting example of an agent inhibiting octameric PBGS is described further below. The octameric form of PBGS binds to substrate in a physiologically relevant concentration range and is active at physiological pH. The octamer is composed of four hugging dimers, where the arms of one subunit hug the barrel of an adjacent subunit with which there are strong barrel-to-barrel interactions. The newly discovered hexameric form of PBGS is an essential component of the regulation of tetrapyrrole biosynthesis in a subset of organisms, including plants and some pathogenic bacteria, but not including humans, animals or fungi. The hexameric form is substantially inactive under physiological conditions. In particular, the hexamer cannot bind substrate in the physiologically relevant concentration range because its K,,, value is at least two orders of magnitude larger than the K,,, of the octamer. The hexamer is composed of three detached dimers, where the N-terminal arms do not interact with the adjacent subunit with which there are strong barrel-to-barrel contacts. The transition between the hexameric form and the octameric form involves a significant change in the protein structure. See, e.g., Fig. 5 A. Certain embodiments ofthe invention relate to the inhibition ofthe structural change from a hexamer to an octamer, to inhibit the activation of PBGS and tetrapyrrole biosynthesis in plants and/or bacteria. Since the inhibition mechanism is effective for plants and bacteria, but not animals, the invention provides a novel approach to bacteriostatic, antibiotic and herbicide applications. Thus, in certain embodiments, the invention comprises an inhibitor ofthe hexamer-to- octamer transition for those PBGS that are physiologically regulated by magnesium. The inhibitor can be a known or novel compound. The inhibitor is effective at inhibiting tetrapyrrole biosynthesis in plants and bacterial pathogens at that point in their growth and development where the hexamer-to-octamer transition is physiologically significant. Inhibition of the quaternary structure transition from hexameric PBGS to octameric PBGS is a novel target for the development of antibiotics and herbicides. There is a phylogenetic variation in PBGS proteins where some have an allosteric magnesium and others do not. The PBGS that have the allosteric magnesium are comprised of the archaea, all the bacteria with the exception of the genus Rhodobacter, and all of the photosynthetic eucarya (e.g., green plants) (24). Another more recent exception appears to be the malaria parasite Plasmodium falciparum. Based on the inventor's previously determined crystal structure for E. coil PBGS and the structure of hexameric PBGS disclosed herein, it appears that the role ofthe allosteric magnesium is to induce a structural change between the low activity hexamer and the high activity octamer. The hexamer-octamer transition for Mg acting on PBGS is a novel structural paradigm for allosteric regulation of protein function. The structure of E. coli PBGS is illustrated in Figs. 9A-C and serves to illustrate the common metal binding variations in PBGS structures. Each E.coli PBGS monomer contains two metal ions, neither of which is phylogenetically conserved. The active site contains a zinc ion that is essential to E.coli PBGS activity but whose three cysteine ligands are not present in many PBGS. This zinc functions in the binding and reactivity ofthe second substrate molecule (33). Details of the zinc site are illustrated in Fig.9B. In addition, there is an allosteric magnesium that is seen bound at the interface of each alpha, beta-barrel with the N-terminal arm of a neighboring subunit; structural details are in Fig.9C. The sequence determinants for binding the allosteric magnesium are not present in all PBGS. Fig. 6 illustrates a schematic for classifying the PBGS into four groups on the basis of whether or not they use an active site zinc and whether or not they use an allosteric magnesium (24). The first matrix (far left) is divided into two classes: (a) active site zinc on the left (shaded), and (b) no active site zinc on the right (unshaded). The second matrix is divided into two classes: (a) no allosteric magnesium on top (diamonds), and (b) allosteric magnesium on the bottom (squares). Combining the two matrixes provides a matrix (far right) consisting of four quadrants, wherein the northwest quadrant (QNW) represents +Zn/-Mg, the northeast quadrant (QNE) represents -Zn/-Mg, the southwest quadrant (QSW) represents +Zn/+Mg, and the southeast quadrant (QSE) represents -Zn/+Mg. The inventor has previously quantified (24) the following distribution of known sequences into the four quadrants: QNW = 9; QNE = 2; QSW = 55 and QSE = 63. Thus, approximately one-half of the currently available sequences encode an active site zinc requirement and one-half do not (i.e., QNW + QSW ~ QNE + QSE). In contrast to the active site metal pattern distribution, more than 90% ofthe PBGS sequences contain the determinants for allosteric magnesium binding (i.e., QSW + QSE » QNW + QNE). The inhibitor will be most effective against a subset of PBGS that contain the allosteric magnesium but do not contain the active site zinc (i.e., PBGS within QSE). These are the photosynthetic eucaryotes and a subset of bacteria, including pathogens such as Pseudomonas aeruginosa. These PBGS proteins elicit the property of protein concentration dependent specific activity, which indicates an interconversion between large active quaternary forms and smaller less active quaternary forms. Also seen for NE quadrant, where preliminary evidence suggests the active form is a hexamer. Thus, in certain preferred embodiments, the inhibitor of the invention is effective to inhibit the formation of octameric PGBS derived from bacteria, archaea, or eucarya, provided that the octameric PGBS contains an allosteric magnesium binding site. A non-limiting list of sources ofthe octameric PGBS, which can be inhibited by the composition ofthe invention, is shown in Fig. 8, which is a classification of organisms including bacteria, archaea and eucarya. Figs. 7A and 7B represent an alignment of active site metal binding residues for the PBGS sequences obtained from GenBank and other web-searchable genomes available as of April 2002. The assignment of an organism into one ofthe four quadrants of Fig. 6 is based on the sequence information presented in Fig.7. The presence of the active site zinc binding site is indicated by a cysteine rich cluster (positions 122, 124, and 132 of human PBGS) in association with an arginine residue on the active site lid (position 221 of human PBGS). Species that do not have the cysteine rich active site zinc binding cluster, contain instead an aspartic acid rich region and the active site lid residue is a lysine In certain embodiments ofthe invention, the inhibitor replaces a metal ion and thereby binds at a metal ion binding site, preferably, the metal ion is zinc or magnesium. In certain embodiments of the invention, the inhibitor binds at an active site. The inhibitor can bind any here, but the binding site must stabilize one quaternary structure. Binding is preferable to a site that is present in one multimer but not the other. Inhibitors ofthe invention can be identified using the following protocol. First, a model is provided for a hexameric form of a PBGS that contains the allosteric magnesium but does not contain the active site zinc. The initial model can, e.g., be one of pea PBGS. Second, small molecule databases are screened in silico for molecules that will fit into a hug-disabling domain adjacent to the N-terminal portion ofthe subunit. The hug-disabling domain is at least one area of the detached dimer on which binding of the inhibitor inhibits the arms of the dimer from hugging the barrel of that dimer which is necessary to form another dimer to form the active octamer. See Fig. 10, wherein circles represent inhibitors. A likely site of a hug-disabling domain is underneath the joint at which a hugging arm joins the body ofthe subunit (i.e., at the "arm-pit"). Theoretically suitable molecules will be empirically tested in vitro by determining their effect on the protein concentration dependent specific activity of pea PBGS, which is available using an artificial gene construct. Those molecules that inhibit the specific activity of the protein in a protein concentration fashion are good inhibitor candidates. The following method will allow identifying inhibitors that will bind anywhere, not necessarily in the hug-disabling domain on PBGS to inhibit octamer formation. A functional assay for specific activity of PBGS will be used first to select potential inhibitors from available molecules that are identified in the computational screen, e.g., substances that are not harmful to humans. After potential inhibitors are selected, they will be further screened for affecting specific activity based on protein concentration. Accordingly, this invention provides a method of affecting a multimeric protein, the method comprising: providing said multimeric protein comprising an assembly having a plurality of units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of said multimeric protein; providing the composition ofthe invention comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to a binding site on the assembly; and contacting the assembly with the agent, wherein the agent affects the equilibrium by binding to the binding site and thereby affecting said multimeric protein. In certain embodiments of the method, affecting said multimeric protein comprises affecting a formation of a quaternary isoform. In certain embodiments of the method, affecting said multimeric protein comprises affecting a function of said multimeric protein. Further provided is a method of inhibiting a multimeric poφhobilinogen synthase from forming an active form, the method comprising: applying the composition ofthe invention to the multimeric poφhobilinogen synthase; associating the composition with the less active form; inhibiting the less active form from assembling into the active form and thereby inhibiting the multimeric poφhobilinogen synthase from forming the active form. A non-limiting example of the inhibitor is a rosemarinic acid or derivatives thereof. A preferred application of the inventive composition is for inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya in a human or an animal host. Other applications of the composition of the invention include prevention or inhibition of biofilms on various surfaces including teeth, pipes, tubing ships, or generally any surfaces immersed in water/air mixtures wherein bacteria causing damage can be found. Thus, for example, the compositions of the invention can be effective to prevent or inhibit growth of barnacles on a surface of a ship. Depending on the targeted organism, a composition of the invention can be used to prevent or inhibit damage caused by certain species. Examples of organisms in QSE in the Table so these organisms are primary targets for applying the composition of the invention. Using Table 1 as a guide, various applications of the composition of the invention can be envisioned such as, for example, a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition and a herbicide. Table 1 Bacteria with PBGS in the SE quadrant
Figure imgf000024_0001
Figure imgf000025_0001
Advantageously, the composition ofthe present invention is effective to cure or prevent a disease caused by bacteria, archaea, and/or eucarya. The composition is effective to prevent formation ofthe multimeric PBGS (e.g., octameric PBGS or another active form having a lesser number of nomomers) and thereby inhibit or prevent development or growth of bacteria, archaea, and/or eucarya. In certain embodiments, the multimeric PBGS contains an allosteric magnesium binding site. In one variant of this embodiment, the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya. In yet another variant of this embodiment, the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide. In certain embodiments, the composition does not contain the allosteric magnesium binding site and the catalytic zinc. In one variant of this embodiment, the composition is effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya. In yet another variant of this embodiment, the composition is at least one of a drug, a toothpaste, a soap, and a disinfectant. Antibiotics, herbicides, and fungicides are often based on the inhibition of an essential pathway that is specific to the bacteria, plant, or fungus and that is not present in humans/animals. For example, 1) the penicillin class of antibiotics is directed against bacterial cell wall biosythesis, and animal cells do not have cell walls, or 2) the herbicide glyphosate is directed against aromatic amino acid biosynthesis, and humans do not have this pathway, we must eat aromatic amino acids. As we learn more about the differences in sequence and structure for various proteins/enzymes, it becomes possible to target an essential pathway that is universally present in animals, plants, bacteria, and fungi. Such is the case for targeting the tetrapyrrole biosynthetic pathway through the inhibition of PBGS as the foundation for antimicrobials or herbicides. The phylogenetic variation in metal binding sites among the PBGS of various organisms provides sufficient structural differences for development of an inhibitory agent that will not be inhibitory toward human PBGS. In the case of PBGS, there are significant differences between organisms in the inherent ability of the PBGS to equilibrate between moφhein forms and in the amino acid sequence of the moφhein surfaces. In the case ofthe more general inhibition of protein function through the selective stabilization of one moφhein form, it may be the case that the target is a pathway that is not present in humans or it may be the case that the target simply has sufficient phylogentic variation outside the active site that the surfaces ofthe moφheins are very different. In certain embodiments, the composition comprises a pharmaceutically-acceptable medium in addition to the agent. The expression "pharmaceutically-acceptable medium" denotes a medium, such as a solvent, that is able to deliver the inhibitor, as well as any other active agents in the composition, to the target organism in a relatively safe and effective manner. The medium itself need not have any pharmaceutical activity. As used herein, "pharmaceutically-acceptable medium" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the inhibitor of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incoφorated into the compositions. Solutions ofthe active ingredients as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent growth of all microorganisms. The compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. The composition ofthe present invention is advantageously administered in the form of iηjectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared. These preparations also may be emulsified. A typical composition for such puφoses comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well know parameters. Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray. An effective amount ofthe therapeutic agent is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity ofthe therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state ofthe subject and the protection desired. Precise amounts ofthe therapeutic composition also depend on the judgment ofthe practitioner and are peculiar to each individual. Another application of the inventive composition is a herbicide, wherein the composition additionally comprises a herbicidally-effective medium. The expression
"herbicidally-effective medium" denotes a medium, such as a solvent, that is able to deliver the inhibitor, as well as any other active agents in the composition, to the target organism. The medium itself need not have any herbicidal activity. Guidance for applying antibacterial compositions on crops is provided as follows: since all photosynthetic eukaryots fall in the QSE quadrant of Fig 6, they are themselves targets for the inhibitors proposed in this invention. However, the arm-pit inhibitor binding site shown in Fig 10 has significant phylogenetic variation between plants and bacteria. Hence, agents that would act as an antibacterial spray on crops would need to be those that will bind to this site in the bacterial PBGS, but not in the plant PBGS. Compositions ofthe present invention include both dilute compositions, which are ready for immediate use, and concentrated compositions, which require to be diluted before use, usually with water. The solid compositions may be in the form of granules, or dusting powders wherein the active ingredient is mixed with a finely divided solid diluent, e.g. kaolin, bentonite, kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth or gypsum or a combination thereof. They may also be in the form of dispersible powders or grains, comprising a wetting agent to facilitate the dispersion ofthe powder or grains in liquid. Solid compositions in the form of a powder may be applied as dusts. Liquid compositions may comprise a solution, suspension, or dispersion ofthe active ingredients in water optionally containing a surface-active agent, or may comprise a solution or dispersion ofthe active ingredient in a water-immiscible organic solvent, which is dispersed as droplets in water. The herbicidal composition is suitable either for tank mixing to produce a dilute composition ready for immediate use or for the formation of a concentrate. The solutions or dispersions may be prepared by dissolving the active ingredients in water or an organic solvent optionally containing wetting or dispersing agent(s) and then, when organic solvents are used, adding the mixture so obtained to water optionally containing wetting or dispersing agent(s). Suitable organic solvents include, for example, ethylene dichloride, isopropyl alcohol, propylene glycol, diacetone alcohol, toluene, kerosene, methylnaphthalene, xylene or trichloroethylene, or a combination thereof. Other additives and adjuvants may also be present in compositions of the present invention. Examples include anti-freeze agents such as ethylene glycol and propylene glycol; dyes; dispersants; rheological agents; anti-foam agents such as silicone based agents; and humectants such as ethylene glycol. Development of herbicide on this basis allows developing herbicide resistant crops by making these resistant crops transgenic (i.e., containing genetic material artificially transferred from another species) for a PBGS that is the top half of the four quadrants of Fig. 6, e.g. human
PBGS. HERBICIDE RESISTANT PLANT Further provided is a herbicide resistant plant adapted to be transgenic for a multimeric poφhobilinogen synthase that substantially exist in a multimeric form of a hugging dimer. In certain embodiments, the multimeric poφhobilinogen synthase is derived from a human. In certain embodiments, the multimeric poφhobilinogen synthase contains no allosteric magnesium binding site. The following provides a guidance to making the herbicide resistant plant adapted to be transgenic for a multimeric poφhobilinogen synthase. The expression in a plant of a gene that exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region, which adds polyadenylate nucleotides to the 3' end ofthe RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complimentary strand of mRNA. This mRNA is then used as a template for the production ofthe protein encoded therein by the cells protein biosynthetic machinery. In the instant invention, the promoter chosen will have the desired tissue and developmental specificity. Therefore, promoter function should be optimized by selecting a promoter with the desired tissue expression capabilities and approximate promoter strength and selecting a transformant that produces the desired PBGS activity. This selection approach from the pool of transformants is routinely employed in expression of heterologous structural genes in plants because there is variation between transformants containing the same heterologous gene due to the site of gene insertion within the plant genome (commonly referred to as "positional effect"). In addition to promoters that are known to cause transcription (constitutively or tissue- specific) of DNA in plant cells, other promoters may be identified for use in the current invention by screening a plant cDNA library for genes that are selectively or preferably expressed during the time of interest and then isolating the promoter regions by methods known in the art. In a preferred embodiment ofthe invention, the PBGS transgene is to be expressed in the chloroplast in response to light. More specifically, the PBGS transgene is transcribed into mRNA in the nucleus and the mRNA is translated into a precursor polypeptide (Chloroplast Transport Peptide (CTPVPBGS) in the cytoplasm. The precursor polypetide is then transported (imported) into the chloroplast. Several chloroplast light inducible promoters that are active in plant cells have been described in the literature. Examples of such promoters include the light-inducible promoter from the small subunit of ribulose-1,5- bisphosphate carboxylase (ssRUBISCO), a very abundant plant polypeptide, the chlorophyll a b binding protein gene promoters and the phytochrome promoter which has been utilized recently in a light-switchable promoter system (Shimizu-Sato et al., 2002). Some of these promoters have been used to create various types of DNA constructs that have been expressed in plants; see, e.g., PCT publication WO 84/02913. Other promoters that are known to or are found to cause transcription of DNA in plant cells in response to light can be used in the present invention. Such promoters may be obtained from a variety of sources such as plants and plant viruses and include, but are not limited to, the enhanced CaMV35S promoter and promoters isolated from plant genes such as small subunit of ribulose- 1 ,5-biphosphate carboxylase (ssRUBISCO) genes. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of PBGS enzyme to produce sufficient tetrapyrroles to sustain growth. In one embodiment, said promoter is leaky in order to provide tetrapyrroles necessary for the non-photosynthetic functions ofthe plant. Plastid-directed Expression of PBGS Activity In a preferred embodiment ofthe invention, the PBGS gene is fused to a CTP, in order to target the PBGS protein to the plastid. As used hereinafter, chloroplast and plastid are intended to include the various forms of plastids including amyloplasts. Many plastid-localized proteins are expressed from nuclear genes as precursors and are targeted to the plastid by a CTP, which is removed during the import steps. Examples of such chloroplast proteins include the small subunit of ribulose-l,5-biphosphate carboxylase (ssRUBISCO, SSU), 5-enolpyruvateshikimate- 3-phosphate synthase (EPSPS), ferredoxin, ferredoxin oxidoreductase, the light-harvesting- complex protein I and protein II, and thioredoxin F. The glyphosate-tolerant EPSP synthase plant gene also encodes a polypeptide which contains a CTP, which enables the EPSP synthase polypeptide to be transported into a chloroplast inside the plant cell (U.S. Patent No. 5310667). It has been demonstrated that non-plastid proteins may be targeted to the chloroplast by use of protein fusions with a CTP and that a CTP sequence is sufficient to target a protein to the plastid. Those skilled in the art will also recognize that various other chimeric constructs can be made that utilize the functionality of a particular plastid transit peptide to import the PBGS enzyme into the plant cell plastid. The PBGS gene could also be targeted to the plastid by transformation of the gene into the chloroplast genome (Daniell et al., 1998). Generally chloroplast uptake signals such as the CTP are rich in Ser, Thr and small hydrophobic amino acid residues. The RNA produced by a DNA construct ofthe present invention may also contain a 5' non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. The present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequence can be derived from an unrelated promoter or coding sequence. In monocots, an intron is preferably included in the gene construct to facilitate or enhance expression ofthe coding sequence. Examples of suitable introns include the HSP70 intron and the rice actin intron, both of which are known in the art. Another suitable intron is the castor bean catalase intron (Suzuki et al., 1994). Polyadenylation Signal The 3' non-translated region ofthe chimeric plant gene contains a polyadenylation signal that functions in plants to cause the addition of polyadenylate nucleotides to the 3' end ofthe
RNA. Examples of suitable 3' regions are (1) the 3' transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean storage protein genes and the small subunit ofthe ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene. Plant Transformation/Regeneration In developing the nucleic acid constructs of this invention, the various components ofthe construct or fragments thereof will normally be inserted into a convenient cloning vector, e.g., a plasmid that is capable of replication in a bacterial host, e.g., E. coli. Numerous vectors exist that have been described in the literature, many of which are commercially available. After each cloning, the cloning vector with the desired insert may be isolated and subjected to further manipulation, such as restriction digestion, insertion of new fragments or nucleotides, ligation, deletion, mutation, resection, etc. so as to tailor the components ofthe desired sequence. Once the construct has been completed, it may then be transferred to an appropriate vector for further manipulation in accordance with the manner of transformation ofthe host cell. A recombinant DNA molecule ofthe invention typically includes a selectable marker so that transformed cells can be easily identified and selected from non-transformed cells. Examples of such include, but are not limited to, a neomycin phosphotransferase (nptll) gene (Potrykus et al., 1985), which confers kanamycin resistance. Cells expressing the nptll gene can be selected using an appropriate antibiotic such as kanamycin or G418. Other commonly used selectable markers include the bar gene, which confers bialaphos resistance; a mutant EPSP synthase gene (Hinchee et al., 1988), which confers glyphosate resistance; a nitrilase gene, which confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS), which confers imidazolinone or sulphonylurea resistance (European Patent Application 154,204, 1985); and a methotrexate resistant DHFR gene (Thillet et al., 1988). Plants that can be made to express the PBGS transgene include, but are not limited to,
Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, Clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon, mushroom, nut, oat, oil seed rape, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, esunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, a vine, watermelon, wheat, yams, and zucchini. A PBGS gene can be inserted into the genome of a plant by any suitable method.
Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella et al. (1983), Bevan (1984), Klee et al. (1985) and EPO publication 120,516. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen. DNA may also be inserted into the chloroplast genome (Daniell et al., 1998).
A plasmid expression vector suitable for the introduction of a PBGS gene in monocots using microprojectile bombardment is composed ofthe following: a CTP; a light inducible promoter; the PBGS gene; an intron that provides a splice site to facilitate expression ofthe gene, such as the Hsp70 intron (PCT Publication WO93/19189); and a 3' polyadenylation sequence such as the nopaline synthase 3' equence (NOS 3'; Fraley et al., 1983). This expression cassette may be assembled on high copy replicons suitable for the production of large quantities of DNA to be injected into the plant. A particularly useful Agrobacterium-based plant transformation vector for use in transformation of dicotyledonous plants is plasmid vector pMON530 (Rogers et al., 1987). Plasmid pMON530 is a derivative of pMON505 prepared by transferring the 2.3 kb Stul-Hindlll fragment of pMON316 (Rogers et al., 1987) into pMON526. Plasmid pMON526 is a simple derivative of pMON505 in which the Smal site is removed by digestion with Xmal, treatment with Klenow polymerase and ligation. Plasmid pMON530 retains all the properties of pMON505 and the CaMV35S-NOS expression cassette and now contains a unique cleavage site for Smal between the promoter and polyadenylation signal. Binary vector pMON505 is a derivative of pMON200 (Rogers et al., 1987) in which the i plasmid homology region, LIH, has been replaced with a 3.8 kb Hindlll to Smal segment of the mini RK2 plasmid, pTJS75 (Schmidhauser and Helinski, 1985). This segment contains the RK2 origin of replication, oriV, and the origin of transfer, oriT, for conjugation into Agrobacterium using the tri-parental mating procedure (Horsch and Klee, 1986). Plasmid pMON505 retains all the important features of pMON200 including the synthetic multi-linker for insertion of desired DNA fragments, the chimeric NOS/NPTII'/NOS gene for kanamycin resistance in plant cells, the spectinomycin/streptomycin resistance determinant for selection in E. coli and A. tumefaciens, an intact nopaline synthase gene for facile scoring of transformants and inheritance in progeny, and a pBR322 origin of replication for ease in making large amounts ofthe vector in E. coli. Plasmid pMON505 contains a single T-DNA border derived from the right end ofthe pTiT37 nopaline-type T-DNA. Southern blot analyses have shown that plasmid pMON505 and any DNA that it carries are integrated into the plant genome, that is, the entire plasmid is the T-DNA that is inserted into the plant genome. One end ofthe integrated DNA is located between the right border sequence and the nopaline synthase gene and the other end is between the border sequence and the pBR322 sequences. Another particularly useful Ti plasmid cassette vector is pMON 17227. This vector is described in PCT Publication WO 92/04449 and contains a gene encoding an enzyme conferring glyphosate resistance (denominated CP4), which is an excellent selection marker gene for many plants, including potato and tomato. The gene is fused to the Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressed from the FMV promoter as described therein. When adequate numbers of cells (or protoplasts) containing the PBGS gene are obtained, the cells (or protoplasts) are regenerated into whole plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for hosts from Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae
(cabbage, radish, canola/rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice, maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), various floral crops, such as sunflower, and nut-bearing trees, such as almonds, cashews, walnuts, and pecans. See, e.g., Ammirato et al. (1984); Shimamoto et al. (1989); Fromm (1990); Vasil et al. (1990); Vasil et al. (1992); Hayashimoto (1990); and Datta et al. (1990). In one embodiment, the PBGS gene is derived from a species in which the PBGS enzyme does not comprise Mg2+ but comprises Zn2+. In a preferred embodiment, the species is yeast or human. In another preferred embodiment, a mutant PBGS gene is used to generate a transgenic plant. In a further embodiment, the PBGS gene is introduced into the plant genome by homologous recombination. The wild type human PBGS genomic DNA and full length cDNA which may be used to generate a transgenic plant are shown below:
Human PBGS gene (SEQ ID NO:l): cttacgcggtctgtgggagaccggagcgggagacagcggtgacaggagcagcggccgggagcccttagggaggcagacagag cctgcagccaatgccccaggagccctcggttccaaccaactgatgcccctgtgcccactggcccacgccatgcagccccagtccgtt ctgcacagcggctacttccacccactacttcgggcctggcagacagccaccaccaccctcaatgcctccaacctcatctaccccatctt tgtcacggatgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtgaagcggctggaagagatgct gaggcccttggtggaagagggcctacgctgtgtcttgatctttggcgtccccagcagagttcccaaggacgagcggggttccgcag ctgactccgaggagtccccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcctgtgatgtctgcctg tgtccctacacctcccatggtcactgcgggctcctgagtgaaaacggagcattccgggctgaggagagccgccagcggctggctga ggtggcattggcgtatgccaaggcaggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatcaaagag gccctgatggcacatggacttggcaacagggtatcggtgatgagctacagtgccaaatttgcttcctgtttctatggccctttccgggat gcagctaagtcaagcccagcttttggggaccgccgctgctaccagctgccccctggagcacgaggcctggctctccgagctgtgga ccgggatgtacgggaaggagctgacatgctcatggtgaagccgggaatgccctacctggacatcgtgcgggaggtaaaggacaag caccctgacctccctctcgccgtgtaccacgtctctggagagtttgccatgctgtggcatggagcccaggccggggcatttgatctcaa ggctgccgtactggaggccatgactgccttccgcagagcaggtgctgacatcatcatcacctactacacaccgcagctgctgcagtg gctgaaggaggaatgatggagacagtgccaggcccaagaactagaactttaaaacgttcccggggcctcagacaagtgaaaacca aagtaaatgctgcttttagaactgtgccctcatgccctcttcctgctcacatgctagcggggcccagcagccctgggtggttttgccagc atgctaactcttgtaactcgcagctgcatcctatgagctctcccaagcttccccgcccctcccctgggtcagccgtgaggcccacctttg ccaccctcagctctttcctctggtgtggcttcagcttgaaagcaacctggagtcgggggcacagcctttggggcctggctgggagag ggtcttggagcattaggggaagaagagagcagtgggatcttggggcctgagaagccttggaacgcttctggcagcagagctgggt gtgggaatgaggcctagatcgatatccctgggttagagttgaaatttgccgcaattccactggaaggcatttcccacgaggccagagg ttgccaggctgcctgaggtctcctattctactctgaaccataaacccagagaagaattactcattaaccagcataaatactgcctgagga tcaaaactcagaggcaaagagggagttcctgactgctagaggtgccaccaccacaaacactttttattcaggagatactttttgagaatc tctgctctgttcctaggttcagtgctgggtcctgggaatacagcaggacagacctcagcttatctcttcatagaaattatacaaagagaat tggggagacagctaagaagaaaacaaagaaataaagcagttacaaattgtgataagtgctttgaaggaaagaaggggtctgagaca acaacagggaaggggcctctcttgaaacagtagttgggaaggaggcagacatgcaccagtgatgtggtgacaggtgctctgaagg aggtcaccaggacctgacctctttgaaggatcagaaaatacttccctgaaggactgacatttgagcctagacctgaagggtgagccat caagctaagacaattggggaagagcattccagggagagggaggagttgtgcaaaggccctggggctccttctagctggaggaatg caaggctagcttgtctggagcactgagaggatggcctgaactgagtggagagagacagaccaggaccaaaccatgcagaggtcaa gggccacattcaccttttcagagtgactcaatcaaatttgtagtttgtaaaagtattttaacagctctgcggcaaagtgcaaatgaaaagtc ttgatggcatggactggagcggggacagtggggatggagaaaggggaatggattgtggatgtgtttagaaggtagattcgatgtgaa ggatgaatctggcttgaccttctgggtggctgatgggccatttactgagatggggcagcctggaagaggaacagaagcagggtcgg ggtggagggagaatactaaacttagcttgagacattttgcaataaggaagctatatctagagtgcttatgtgactcacctaaggccactc aacaagtttgtggcagaactggattagaactgcacagaaaacagccaagctgggatttgaacccatgtagtccaactccaaggcctct gcccctaaccactgtgccataccacctcccaataatcaacagcaaaattataggtctaacaatgttttatagacacccctccatttatgtga tgggtttgcatcctgataaacccatcataagttgaaaatatgatcataagttgaaaatatgatcataagtcaaaaatgtatttaatataccta acctaccaaacatcatagcttagcctagcctgccttaaacatgctcagaacacttacattagcctacagtgggcaaaactatccaacaca aaatctatattgtaataaagttgtaaagaattttgaataaaaattcaatatttgaaaaaaaaaaaaaaaaa
Human PBGS cDNA (SEQ ID NO:2): gcagccaaagccccaggagccctaggttccaaccaactgatgcccctgtgcccactggcccacgccatgcagccccagtccgttctgca cagcggctacttccacccactacttcgggcctggcagacagccaccaccaccctcaatgcctccaacctcatctaccccatctttgtcacgg atgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtgaagcggctggaagagatgctgaggcccttggt ggaagagggcctacgctgtgtcttgatctttggcgtccccagcagagttcccaaggacgagcggggttccgcagctgactccgaggagtc cccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcctgtgatgtctgcctgtgtccctacacctcccatggt cactgcgggctcctgagtgaaaacggagcattccgggctgaggagagccgccagcggctggctgaggtggcattggcgtatgccaagg caggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatcaaagaggccctgatggcacatggacttggcaac agggtatcggtgatgagctacagtgccaaatttgcttcctgtttctatggccctttccgggatgcagctaagtcaagcccagcttttggggacc gccgctgctaccagctgccccctggagcacgaggcctggctctccgagctgtggaccgggatgtacgggaaggagctgacatgctcatg gtgaagccgggaatgccctacctggacatcgtgcgggaggtaaaggacaagcaccctgacctccctctcgccgtgtaccacgtctctgga gagtttgccatgctgtggcatggagcccaggccggggcatttgatctcaaggctgccgtactggaggccatgactgccttccgcagagca ggtgctgacatcatcatcacctactacacaccgcagctgctgcagtggctgaaggaggaatgatggaggacagtgccaggcccaagaac tagaactttcaaacgttcccggggcctcagacaagtgacaaccaaagtaaatgctgcttttagaactgt
Human PBGS amino acid sequence (SEQ ID NO:3):
MQPQSVLHSGYFHPLLRAWQTATTTLNASNLIYPIFVTDVPDDIQPITSLPGVARYGV KRLEEMLRPLVEEGLRCVLIFGVPSRVPKDERGSAADSEESPAIEAIHLLRKTFPNLL VACDVCLCPYTSHGHCGLLSENGAFRAEESRQRLAEVALAYAKAGCQVVAPSDM
MDGRVEAIKEALMAHGLGNRVSVMSYSAKFASCFYGPFRDAAKSSPAFGDRRCYQ LPPGARGLALRAVDRDVREGADMLMVKPGMPYLDIVREVKDKHPDLPLAVYHVS GEFAMLWHGAQAGAFDLKAAVLEAMTAFRRAGADIIITYYTPQLLQWLKEE The compositions of the invention is suitable as antimicrobial active ingredients in personal care preparations, for example shampoos, bath additives, hair-care products, liquid and solid soaps (based on synthetic surfactants and salts of saturated and/or unsaturated fatty acids), lotions and creams, deodorants, other aqueous or alcoholic solutions, e.g. cleansing solutions for the skin, moist cleansing cloths, oils or powders. The invention therefore relates also to a personal care preparation comprising the composition of the invention and optionally cosmetically tolerable carriers or adjuvants as described in U.S. Patent No.6,689,372 to Holzl et al. The composition are to be used in amounts effective to have the antimicrobial effect, i.e. inhibit or prevent microbial activity. Other constituents can be used, for example sequestering agents, colourings, perfume oils, thickening or solidifying (consistency regulator) agents, emollients, UV absorbers, skin-protective agents, antioxidants, additives that improve mechanical properties, such as dicarboxylic acids and/or Al, Zn, Ca and Mg salts of fatty acids, and optionally preservatives. Further, the invention provides a method of antimicrobial treatment of skin, mucosa or hair which comprises, contacting the surface ofthe skin, mucosa or hair of a person in need of said antimicrobial treatment with an antimicrobially effective amount ofthe compound ofthe invention. The personal care preparation according to the invention may be formulated as a water- in-oil or oil-in- water emulsion, as an alcoholic or alcohol-containing formulation, as a vesicular dispersion of an ionic or non-ionic amphiphilic lipid, as a gel, a solid stick or as an aerosol formulation. As a water-in-oil or oil-in-water emulsion, the cosmetically tolerable adjuvant contains, for example, from 5 to 50% of an oily phase, from 5 to 20% of an emulsifier and from 30 to 90% water. The oily phase may contain any oil suitable for cosmetic formulations, e.g. one or more hydrocarbon oils, a wax, a natural oil, a silicone oil, a fatty acid ester or a fatty alcohol. Preferred mono- or poly-ols are ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and sorbitol. Cosmetic formulations according to the invention may be contained in a wide variety of cosmetic preparations as described in U.S. Patent No. 6,689,372 to Holzl et al. Especially the following preparations, for example, come into consideration: skin-care preparations, e.g. skin- washing and cleansing preparations in the form of tablet-form or liquid soaps, soapless detergents or washing pastes; bath preparations, e.g. liquid (foam baths, milks, shower preparations) or solid bath preparations, e.g. bath cubes and bath salts; skin-care preparations, e.g. skin emulsions, multi-emulsions or skin oils; cosmetic personal care preparations, e.g. facial make-up in the form of day creams or powder creams, face powder (loose or pressed), rouge or cream make-up, eye-care preparations, e.g. eyeshadow preparations, mascara, eyeliner, eye creams or eye-fix creams; lip-care preparations, e.g. lipsticks, lip gloss, lip contour pencils, nail- care preparations, such as nail varnish, nail varnish removers, nail hardeners or cuticle removers; intimate hygiene preparations, e.g. intimate washing lotions or intimate sprays; foot-care preparations, e.g. foot baths, foot powders, foot creams or foot balsams, special deodorants and antiperspirants or callous-removing preparations; light-protective preparations, such as sun milks, lotions, creams and oils, sun blocks or tropicals, pre-tanning preparations or after-sun preparations; skin-tanning preparations, e.g. self-tanning creams; depigmenting preparations, e.g. preparations for bleaching the skin or skin-lightening preparations;insect-repellents, e.g. insect-repellent oils, lotions, sprays or sticks; deodorants, such as deodorant sprays, pump-action sprays, deodorant gels, sticks or roll-ons; antiperspirants, e.g. antiperspirant sticks, creams or roll-ons; preparations for cleansing and caring for blemished skin, e.g. soapless detergents (solid or liquid), peeling or scrub preparations or peeling masks; hair-removal preparations in chemical form (depilation), e.g. hair-removing powders, liquid hair-removing preparations, cream- or paste-form hair-removing preparations, hair-removing preparations in gel form or aerosol foams; shaving preparations, e.g. shaving soap, foaming shaving creams, non-foaming shaving creams, foams and gels, preshave preparations for dry shaving, aftershaves or after-shave lotions; fragrance preparations, e.g. fragrances (eau de Cologne, eau de toilette, eau de parfum, parfum de toilette, perfume), perfume oils or cream perfumes; dental-care, denture-care and mouth-care preparations, e.g. toothpastes, gel tooth-pastes, tooth powders, mouthwash concentrates, anti -plaque mouthwashes, denture cleaners or denture fixatives; cosmetic hair- treatment preparations, e.g. hair-washing preparations in the form of shampoos and conditioners, hair-care preparations, e.g. pretreatment preparations, hair tonics, styling creams, styling gels, pomades, hair rinses, treatment packs, intensive hair treatments, hair-structuring preparations, e.g. hair-waving preparations for permanent waves (hot wave, mild wave, cold wave), hair- straightening preparations, liquid hair-setting preparations, foams, hairsprays, bleaching preparations; e.g. hydrogen peroxide solutions, lightening shampoos, bleaching creams,
^bleaching powders, bleaching^pastes or oils, temporary, semj-permanenfor permanent hair colourants, preparations containing self-oxidising dyes, or natural hair colourants, such as henna or camomile. The oral composition according to the invention may be, for example, in the form of a gel, a paste, a cream or an aqueous preparation (mouthwash). The oral composition according to the invention may also comprise compounds that release fluoride ions which are effective against the formation of caries, for example inorganic fluoride salts, e.g. sodium, potassium, ammonium or calcium fluoride, or organic fluoride salts, e.g. amine fluorides, which are known under the trade name Olafluor. The compositions ofthe invention are also suitable for the treatment of textile fibre materials. Such materials are undyed and dyed or printed fibre materials, e.g. of silk, wool, polyamide or polyurethanes, and especially cellulosic fibre materials of all kinds. Such fibre materials are, for example, natural cellulose fibres, such as cotton, linen, jute and hemp, as well as cellulose and regenerated cellulose. Preferred suitable textile fibre materials are made of cotton. The compositions of the invention can also be used in washing and cleaning formulations, e.g. in liquid or powder washing agents or softeners. The compositions of the invention are also suitable for imparting anti-microbial properties to plastics, e.g. polyethylene, polypropylene, polyurethane, polyester, polyamide, polycarbonate, latex etc. Fields of use therefor are, for example, floor coverings, plastics coatings, plastics container and packaging materials, kitchen and bathroom utensils (e.g. brushes, shower curtains, sponges, bathmats), latex filter materials (air and water filters), plastics articles used in the field of medicine, e.g. dressing materials, syringes, catheters etc., so- called "medical devices", gloves and mattresses. Paper, for example papers used for hygiene puφoses, may also be provided with antimicrobial properties using the compositions according to the invention. It is also possible for nonwovens, e.g. nappies/diapers, sanitary towels, panty liners, and cloths for hygiene and household uses, to be provided with antimicrobial properties in accordance with the invention. The compositions can be used especially also in household and all-puφose cleaners for cleaning and disinfecting hard surfaces. In addition to preserving cosmetic and household products, technical products, such as paper treatment liquors, printing thickeners of starch or of cellulose derivatives, surface-coatings and paints, can be preserved and provided with antimicrobial properties. The compositions ofthe invention are also suitable for the antimicrobial treatment of wood and for the antimicrobial treatment of leather and the provision of leather with antimicrobial properties. The compounds according to the invention are also suitable for the protection of cosmetic products and household products from microbial damage Further, the composition ofthe present invention can be used as an oral composition such as a dentifrice composition in association with an orally-acceptable carrier as described in U.S. Patent No. 6,740,311 to White, Jr., et al. Non-limiting examples of such oral composition are toothpastes, tooth powders, prophylaxis pastes, lozenges, gums and the like suitable for humans and animals. Further, the compositions ofthe invention can be used to prepare antimicrobial surfaces.
Further provided is a method of making an antibacterial surface, the method comprising: (1) providing the composition of the invention wherein the composition is effective to inhibit or prevent formation ofthe active form ofthe multimeric poφhobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric poφhobilinogen synthase contains an allosteric magnesium binding site and the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide; (2) providing a surface-forming matrix; and (3) combining the composition with the surface-forming matrix and thereby making the antibacterial surface. In one variant of the method, the antibacterial surface is adapted to prevent or inhibit a formation of a biofilm. The term "a surface-forming matrix" as used herein includes polymers, biodegradable and no-biodegradabe, silicas, ceramics and combinations thereof for mixing, layering or otherwise associating the composition with the matrix. The composition can also be put on the top or a bottom surface ofthe matrix. In this invention, provided is a method for manipulating growth or development of a plant comprising applying the composition of the invention which is a herbicide to the plant, wherein the plant is herbicide resistant and is adapted to be transgenic for a multimeric porphobilinogen synthase that substantially exist in a multimeric form of a hugging dimer. In one variant of the method, the multimeric porphobilinogen synthase contains no allosteric magnesium binding site. The invention will be illustrated in more detail with reference to the following Example, but it should be understood that the present invention is not deemed to be limited thereto. EXAMPLE PORPHOBILINOGEN SYNTHASE AS AN EXAMPLE OR MORPHEINS: The following example describes the discovery that PBGS can exist in alternate quaternary states and that the interconversion of these states forms the structural basis for allosteric regulation of this PBGS in some species. The well known quaternary state for PBGS is the octamer, made up of hugging dimers. Also known was that some PBGS, particularly those in the QSE of figure 6, exist as an equilibrium of quaternary forms as shown by a protein concentration dependence to the specific activity. The protein concentration dependence to the specific activity indicates that a maximally active oligomer can dissociate or reassociate into smaller less active forms. It was previously believed that the smaller, less active forms, were also multiplicities of hugging dimers. Observation and characterization of a stable oligomer of detached dimers was made possible by the fact that PBGS in the QNW do not readily equilibrate between quaternary isoforms. Hence the F 12L mutation of human PBGS allowed us to study a stable form of the hexamer and to establish that it was the hexameric property (and not the specific F12L mutation) that dictated the dramatically different functional properties of F12L relative to wild type human PBGS. F 12L is naturally occurring rare allele for human PBGS (3- 5). Described below are studies of human PBGS (both wild type and F12L0 that were heterologously expressed in E. coli and purified by conventional techniques. PROTEIN EXPRESSION The parent human PBGS is the well-characterized N59/C 162A(6). N59 corresponds to the more soluble of two codominant alleles encoding the PBGS protein. C162A is a benign mutation that removes the possibility of a slowly forming aberrant disulfide bond. The artificial gene for N59/C162A is called Wt below. The sense strand primer used for the QuikChange mutagenesis of Wt to the F12L variant was GGCTACCTCCACCCACTGCTTCGGGCC. Several constructs were prepared for the coexpression of Wt and F12L in E. coli. Both the order ofthe genes and the number of promoters were varied, but these variations did not affect the outcome. The construct containing Wt and F 12L under the control of one promoter is described. Plasmid DNA containing Wt (pET3aWt) was digested with BamHl and Ndel to cut out Wt.
The pETl 7b vector DNA was linearized by digestion with BamHl and Ndel and ligated with Wt such that the ATG start codon of Wt was 6 basepairs downstream ofthe ribosomal binding site encoded by the vector. The resultant plasmid was transformed into E. coli XL 1 blue. Plasmid DNA (pET17bWt) was prepared and linearized with Spel and Bpul l021. Plasmid DNA containing the gene F12L (pET3aF12L) was digested with Xbal and Bpul 1021 to produce a fragment containing the ribosomal binding site and the gene for F12L. The gene for F12L and the linearized pET17Wt vector were ligated such that the ribosomal binding site of the F12L gene was 35 basepairs downstream ofthe stopcodon of Wt, and the terminator was 52 basepairs downstream ofthe stop codon for the F12L gene. Plasmid pET17bWtF12L was transformed into E.coli XL 1 blue, plasmid DNA was prepared and transformed into E. coli BLR(DE3) for protein expression as previously described (6). PROTEIN PURIFICATION The bulk of the protein purification procedure (cell disruption, ammonium sulfate fractionation, hydrophobic chromatography on Phenyl-Sepharose, anion exchange chromatography, and gel filtration chromatography on Sephacryl S-300) followed the procedures previously described (6) with the exception that a 70 ml Q-Sepharose column was used in place ofthe DEAE agarose column for the anion exchange step. The Q-Sepharose was run at room temperature using 30 mM potassium phosphate, pH 7.0, 10 mM 2-mercaptoethanol, 10 μM Zn(II), and employed a KC1 gradient as shown in Fig. 3 A. The gradient was controlled by a Rainin HPLC system run at a flow rate of 3 ml min'l and 10 ml fractions were collected. KINETIC CHARACTERIZATION OF THE PBGS VARIANTS WAS USED TO SHOW THAT WT AND F12L HAVE DIFFERENT FUNCTIONAL CHARACTERISTIC: All kinetic determinations were carried out in 0.1 M bis-tris propane, 10 mM
2-mercaptoefhanol, 10 μM Zn. For the pH rate profiles, the reported pH reflects the assay pH after the addition of 10 mM ALA-HCl. For Km and Vmax determinations, concentrations of
ALA were 10 μM, 30 μM, 100 μM, 300 μM, 1 mM, 3 mM, and 10 mM and were each done in duplicate. Variations in the concentration of ALA-HCl did not lead to variations in final pH because the stock 0.1 M ALA-HCl was diluted into 0.1 M HCl prior to addition of a constant volume to the assay mixture. All assays were at 37°C for a fixed time using Ehrlich's reagent to determine poφhobilinogen formed.
ANALYTICAL ULTRACENTRIFUGATION: Protein samples were dialyzed into 30 mM potassium phosphate, pH 7.5, 0.1 mM DTT, and 10 μM ZnCl2 just prior to loading into the ultracentrifuge. Loading concentrations were
10.6 μM and 12.8 μM for wild type and F12L mutant enzyme, respectively. All sedimentation equilibrium experiments were carried out at 4°C using a Beckman Optima XL-A analytical ultracentrifuge equipped with an An60 Ti rotor and using six-channel, 12-mm path length, charcoal-filled Epon centeφieces using quartz windows. Data were collected at three rotor speeds (8,000, 11,000, and 14,000 φm) and represent the average of 20 scans using a scan step size of 0.001 cm. Temperature-corrected partial specific volumes and solution density were calculated using the Sednteφ program (32); the solution density was 1.00191 gm/mL and the partial specific volumes were 0.7394 and 0.7397 mL/gm for the wild type and mutant proteins respectively. Data were analyzed using the HID program from the Analytical Ultracentrifugation Facility at the University of Connecticut (Storrs, CT). Model analysis ofthe data ruled out a single species as the residuals from the fits were clearly nonrandom. A CRYSTAL STRUCTURE WAS DETERMINED FOR F 12 : F12L was dialyzed against 50 mM bis-tris propane, 10 mM βME, and 10 μμM ZnCl2.
Crystals were formed using the sitting drop method, equal volume of F12L (4.0 mg ml"1) was mixed with the precipitant (0.4 M monoammonium hydrogen phosphate). ALA was added equimolar to the protein subunit concentration and crystals formed in 3-5 days. Diffraction data were collected at 100K on MAR345 image plate detector coupled with RU-200 rotating anode generator equipped with OSMIC optics and operated at 50kV and lOOma. Crystals were cryoprotected before freezing by transferring them at reservoir solutions containing 12%>, 17%, 23%o and 30%> glycerol for 3 min in each solution. A few data sets were collected showing high degree of disorder and lack of any ligand in the active site area. Because of that, a crystal of F12L was soaked in 2 mM ALA, which was added to the first two cryoprotectant solutions and 0.2 mM ZnCl2, which was added to the last two solutions in addition to ALA. The final data set consisted of 525 frames corresponding to 0.5° oscillation with exposure time 3.5 min per frame. Crystals belong to a hexagonal system, space group P63, unit cell parameters α=b=89.6 A, c=153.2 A. There are two molecules in the asymmetric unit. Diffraction data were reduced with the program package HKL2000, Rmerge(I)=5.0% for 33,615 reflections for the 45-2.2 A resolution range. The structure was solved by molecular replacement with the AmoRe program package by using molecule A of human PBGS structure (pdb code 1E51) as an initial model. Refinement was carried out with program CNS . The final model included one dimer of F 12L - molecule A (residues 11-82, 97-124, 140-169, 172-212, 222-330) and B (residues 3-82, 97- 122, 140-169, 172-212, 226-328), one molecule of an intermediate product ofthe catalytic reaction bound in the active site of molecule A, 241 water molecules and two atoms of Zn which appear to have low occupancies. The crystallographic R-factor is 19.9%, R(free) is 28.6% for 2.2A resolution data, and the RMS deviations for bond lengths and bond angles are 0.18A and 2.0°, respectively. All residues belong to allowed conformation regions on the Ramachandran plot. THE PROPERTIES OF HUMAN PBGS VARIANT F12L Human PBGS variant F12L is remarkably different from the wild type protein. Characterization of purified F12L confirmed that the catalytic activity is very low under conditions where wild type human PBGS is most active. However, F 12L exhibits a remarkably altered pH rate profile and shows considerable activity at basic pH values (Fig. 1 A). The Km and Vmax values of F12L and wild type human PBGS were determined at pH 7, which is optimal for the wild type protein, and at pH 9, which is optimal for F12L; the results are presented in Table 1 (below). F12L exhibits normal Michaelis-Menten kinetics with extraordinarily high Km values, well above physiological concentrations of the substrate 5- aminolevulinic acid (ALA). However, at pH 9 the Vmax of F12L is significantly higher than that ofthe wild type protein. Under conditions of optimal pH and in the presence of an optimal configuration of metal ions, wild type PBGS from all species characterized are reported to have Km values in the range of 100 μM (6, 8-10), as is seen here for wild type human PBGS at pH 7. The kinetic behavior of wild type human PBGS at pH 9 did not exhibit standard Michaelis-
Menten kinetics, the basis of which was not at first apparent. On cursory examination, the wild type protein appeared to exhibit an extreme negative cooperativity with a Hill coefficient on the order of 0.35. In fact, the best fit for the data was to a double hyperbolic equation, which was later appreciated to derive from catalysis by a mixture of quaternary isoforms (moφheins, octamer and hexamer) where the two forms have very different Km values. This phenomenon is described in more detail below. Further evidence for extraordinary differences between the F12L variant and the wild type protein came from variations in mobility during anion exchange chromatography (Fig. 1 B) and during native gel electrophoresis (Fig. 1 C), both of which suggest a difference in oligomeric structure. Separation on an anion exchange column generally reflects a different surface charge, which cannot be due to the replacement of neutral leucine for neutral phenylalanine. Separation of two species with identical charge/mass ratio by electrophoresis indicates either a different size or a different shape. Together, these differences suggested that F 12L and wild type human
PBGS exist in different oligomeric states. When the wild type and mutant proteins were subjected to sedimentation equilibrium analysis using an analytical ultracentrifuge, the molecular weight for the wild type protein and F 12L were found to be 244,000 ± 8,900 and 197,900 ± 6,500 Daltons, respectively. The former is midway between that expected for an octamer and a hexamer, while the latter is midway between that expected for a hexamer and a tetramer. In model analysis ofthe data, the wild type protein fit best to a three-state model of dimer, hexamer, and octamer at 7.6%, 51%, and 42% respectively, while F 12L fit best to a two-state model of tetramer and hexamer at a ratio of 70%> to 30%>, with octamer absent. Hence the inventor undertook the determination of the crystal structure of human PBGS variant F 12L.
THE CRYSTAL STRUCTURE OF HUMAN PBGS AND THE F12L VARIANT SHOW REMARKABLE DIFFERENCES IN THE STRUCTURE OF THE MONOMER, WHICH DICTATES A NEW QUATERNARY ISOFORM AND REVEALS THE FIRST EXAMPLE OF MORPHEINS: Seventeen previously determined crystal structures of PBGS (1.1-20) from fungi, metazoa, and bacteria reveal a common homo-octameric structure in which four dimers are related by a 90° rotation around a central axis (Fig. 2A). PBGS is a member of the aldolase superfamily of TIM α/β barrel proteins (21). In each subunit the catalytic core resides completely within the barrel and a 20+ amino acid N-terminal arm is involved in extensive subunit interactions. The sequence ofthe catalytic core is phylogenetically conserved, but that of the N-terminal arm is not. The PBGS dimer seen in the octamer (Fig. 2A, top) involves highly conserved barrel-to-barrel contacts and the N-terminal arm of one subunit is hugging the barrel ofthe sister subunit. Hence, this has been referred to as the hugging dimer (2). The side chain of amino acid 12 does not participate in the hugging interaction. Assembly ofthe tetramer, which is by addition of a second hugging dimer rotated 90° around the central axis (Fig. 2A, middle), adds an additional reciprocal interaction between the arm of one subunit and the base of an α/β barrel from a neighboring dimer. The side chain of amino acid 12 participates in this subunit interaction. Addition of two more dimers, each rotated 90° around the central axis results in the octamer (Fig 2 A, bottom). The octamer, rotated 90° toward the reader relative to the view of the dimer and tetramer, and gives a pinwheel representation. Prior to the determination ofthe crystal structure of F12L, it was presumed that all PBGS proteins shared the same homo-octameric structure (2). However, for PBGS from green plants and some bacteria, there is kinetic evidence suggesting that the maximally active octamer can dissociate into smaller, less active, structural units (9, 22). This kinetic evidence is a protein concentration to the specific activity as illustrated in Fig X for pea PBGS. Strikingly, the newly determined crystal structure ofthe F 12L human PBGS allele (PDB
Code 1PV8) reveals a quaternary structure that involves significant rearrangement of the N-terminal arm relative to the α/β barrel (Fig. 2B). In this case, the dimer retains the aforementioned barrel-to-barrel contacts but the N-terminal arms are detached rather than hugging (Fig. 2B, top). Assembly of the tetramer retains the aforementioned reciprocal interaction between the arm of one subunit and the base of an α/β barrel from a neighboring dimer. However, because the arm is jutting out, this association dictates a 120° rotation around the central axis. Hence, in the oligomeric structure there are three detached-dimers, each rotated 120° around a central axis to form a hexamer (Fig. 2B, bottom, viewed in the pinwheel representation). The unprecedented structural transition from the octamer observed for wild type human PBGS to the hexamer observed for F 12L is an outstanding example of how a small mutational change can have a profound effect on the structure and function of a protein and indicates how close in energy these two quaternary forms are. It is also clear from viewing these structures that any equilibration between octamer and hexamer must proceed through the interconversion ofthe hugging dimer and the detached dimer. This interconversion process is illustrated in Fig. 5 A. The new structure of F 12L (2.2 A resolution) contains significant regions of disorder that impede a structural comparison ofthe active site relative to the previously deposited wild type human PBGS structure (PDB code 1E51, 2.83 A resolution). Amino acid 12 does not interact directly with active site residues in either structure. Furthermore, for those amino acids observed in both structures, most are superimposable. Thus, to further probe the basis for the unusual kinetic properties of F12L (e.g., Fig. 1 , Table 1), the inventor undertook coexpression of F12L and wild type human PBGS. COEXPRESSION OF WILD TYPE HUMAN PBGS AND F12L REVEALED THAT THE QUATERNARY STRUCTURE IS THE BASIS FOR THE KINETIC DIFFERENCES: A coexpression system was prepared to produce both wild type human PBGS and the
F12L variant in a 1 :1 ratio from the same RNA message. Purification of the co-expressed protein, called WT+F12L, was found to yield two distinct peaks of PBGS protein on anion exchange chromatography (Fig. 3 A). The peak to elute first (Pool I) runs comparably to F12L on a native gel, while the second peak (Pool II) runs comparably to wild type human PBGS
(Fig. 3B). Pool I showed enhanced activity at pH 9 and Pool II showed enhanced activity at pH
7 (Fig. 3C). Both pools were individually subjected to analysis by mass spectroscopy following a tryptic digest and each was found to contain significant amounts of both the N-terminal 2010.2
Dalton Phe-containing peptide and the 1976.2 Dalton Leu-containing peptide, confirming that both pools contain heteromeric species. The percentage of each chain in the heteromeric pools was quantified by N-terminal sequencing to show that the Pool I contains 48.5% Phe and 51.5%> Leu while Pool II contains 71.1% Phe and 28.3% Leu. These ratios do not obviously reveal what governs the quaternary structure ofthe heteromeric species. Pools I and II were further purified by gel filtration on Sephacryl S300, which reduced cross contamination of the heteromers. The pH rate profiles ofthe S300 purified Pools I and II are remarkably like F12L and wild type human PBGS, respectively (Fig. 3C). Based on the chromatographic, mass spectroscopy, and quantitative N-terminal sequencing data, we conclude that Pool I is comprised of heterohexamers and that Pool II is comprised of heterooctamers. The pH rate profiles are found to be dominated more by the quaternary structure than by the amino acid composition at position 12. The kinetic parameters Km and Vmax ofthe S300 purified pools were determined at pH
7 and at pH 9 (Table 2). The kinetic data do not follow a simple Michaelis-Menten relationship (hyperbolic fit), but can be attributed to catalysis by two different forms ofthe enzyme that have different Km and Vmax values (double hyperbolic fit) (23). Fig. 3D shows activity as a function of substrate concentration; the kinetic data uniformly fit a model where hexameric and octameric forms ofthe enzyme exhibited high and low Km values, respectively. This double hyperbolic fit (dark lines) is far superior to the single hyperbolic fit (light lines). With the exception of the trace amount of octamer present for Pool I that is detected at pH 9, all the kinetic values are well determined (see Table 2). The data for wild type human PBGS at pH 9 also provided a superior fit to the octamer-hexamer model and this solution is included in Table 2. The factors that govern equilibration of human PBGS heteromers under assay conditions remain to be elucidated. Table 2 The kinetic parameters for wild type human PBGS, F 12L, and the pools of heteromeric Wt/F12L
Figure imgf000046_0001
Pool I and Pool II are the two pools of PBGS activity eluted from the Q-Sepharose column, as illustrated in Fig. 3A, and following further purification on a Sephacryl S-300 column. Kmι and Km2 (both mM) are inteφreted as the Km for the octamer and hexamer, respectively. The reported Vmax values (in units of μmoles h"l mg"1) reflect the mole fraction of quaternary species under assay conditions, which remains to be determined. Fitted Km values are independent ofthe distribution of quaternary species. The data presented on wild type human PBGS, the F12L variant, and the WT+F12L heteromers definitively establishes that the kinetic differences between the wild type protein and the F12L variant are primarily due to the difference in quaternary structure. Further work on other select human PBGS mutants (R240A, T23P, and T23P/F 12L) confirm that the kinetics of the hexamer are like the kinetics of F12L and that the kinetics ofthe octamer are like that ofthe wild type protein. In light ofthe structures of octameric vs. hexameric human PBGS, a hypothesis can be formulated concerning the dramatic difference in pH optimum for these two forms of PBGS. The chemistry ofthe PBGS catalyzed reaction requires the formation of at least two Schiff base intermediates (2, 12, 16, 17, 20). Formation of the carbinolamine precursors to these Schiff bases requires that the participating amino groups are uncharged, or that the local pH is above the pKa of the amino groups. One significant structural difference between hexameric and octameric PBGS is the degree of order found in the amino acids that comprise the active site lid. The crystal structure of hexameric PBGS F12L is lacking in density from most ofthe residues that make up the active site lid, thus implying that the hexamer structure destabilizes the closed lid configuration. In the absence of a closed lid to isolate the active site from bulk solvent, the PBGS catalyzed reaction cannot proceed until the external pH is above the pKa ofthe amino groups that participate in Schiff base formation. Hence, the hexameric structure is proposed to exhibit activity only when the external pH is sufficiently basic to facilitate Schiff base formation. The high Km can also be attributed to destabilization of the active site lid since crystal structures ofthe PBGS octamer show stabilizing interactions between residues on the lid and the substrate molecule that determines the Km value. The current results provide a novel approach to understanding the regulation of PBGS function. As described below, the insight provided from identification of a PBGS hexamer has considerable significance for rethinking the allosteric regulation of PBGS activity in non-human species.
ALLOSTERIC REGULATION OF PBGS CAN BE ATTRIBUTED TO THE OCTAMER TO HEXAMER EQUILIBRIUM: Comparison ofthe PBGS octamer and hexamer reveals a basis for allosteric regulation of PBGS. Despite the fact that all the obvious components of the PBGS active site are contained in the monomer, most PBGS proteins contain a binding site for an allosteric magnesium that is located at the arm-to-barrel interface of the hugging dimer (14, 24). The position of the allosteric magnesium is seen in the crystal structures of both Pseudomonas aeruginosa (14) and E. coli PBGS (16), as illustrated for the latter in Fig.4A. Fig.4 A shows the hugging dimer (light ribbon, dark strand) with the allosteric magnesium as black balls, one of which is illustrated with a large white-on-black arrow. The structures of yeast and human PBGS show that the guanidinium group of an arginine resides in the place ofthe allosteric magnesium as illustrated previously (2). This is Arg240 of human PBGS. If one presumes that all PBGS can exist in the hexameric state under appropriate conditions, then the position ofthe allosteric magnesium is pertinent to a hexamer-octamer transition because this metal binding site is present in the octamer (made up of hugging dimers) and absent in the hexamer (made up of detached dimers). Fig. 4B shows the three subunit to subunit interfaces in the PBGS octamer. The black-on-white arrow shows the barrel-to-barrel interface, which is common to both octameric and hexameric PBGS assemblies. The dots-on-black arrow shows the arm-to-base-of- barrel interaction, which is also common to both octameric and hexameric PBGS assemblies.
The white-on-black arrow, which is analogous to the allosteric magnesium binding site, shows the arm to barrel interaction that is present in the octamer (hugging dimer) and absent in the hexamer (detached dimer). Consistent with the notion that the allosteric magnesium mediates a hexamer-octamer equilibrium is the effect of magnesium on the kinetic parameters of E. coli PBGS. In this case, the addition ofthe allosteric magnesium causes the Km value to decrease from -2 mM to -200 Mm (8), which is remarkably reminiscent ofthe difference between the Km values ofthe hexameric and octameric forms of human PBGS (Table 1). Also of note is our prior observation that homogeneously pure E. coli PBGS shows multiple bands during native gel electrophoresis, that the mobility of these bands is consistent with the molecular size of octamer, hexamer, and dimer, and that addition of magnesium favors the largest (octameric) form (8). Also of note is the recent finding that human PBGS variant R240A purifies -80 % as the hexamer and 20%> as the octamer, and that the latter oligomer is unstable and rearranges to the hexamer with time. OBSERVATION OF PROTEIN CONCENTRATION-DEPENDENT SPECIFIC ACTIVITY IS
THE MOST DIRECT DIAGNOSTIC TOOL FOR THE PRESENCE OF AN EQUILIBRIUM OF MORPHEINS: Interconversion of PBGS between hexamer and octamer is proposed as the mechanism responsible for the protein concentration-dependent specific activity of PBGS from some species. To date we have characterized four different PBGS that contain the allosteric magnesium. The enzymes are from the species E. coli (a γ-proteobacter), B. jαponicum (an α-proteobacter), P. aeruginosa (a γ-proteobacter), and Pisum sativum (a green plant). The last three are different from human PBGS in that they do not use an active site catalytic zinc (24) and they also share the unusual property of protein concentration dependent specific activity (9,
22, 25). The latter property indicates that a maximally active oligomer can dissociate into less active or inactive smaller forms. Published mathematical models have considered maximally active octamers dissociating into less active or inactive tetramers and/or dimers (9, 22). The hexameric structure of human PBGS variant F12L leads us to propose that the protein concentration dependence of plant and certain bacterial PBGS is rather due to an equilibrium between a less active hexameric form and a more active octameric form, as illustrated in Fig. 5A. The existence of such an equilibrium is supported by sedimentation equilibrium studies on pea PBGS (unpublished data). Because magnesium is integral to the difference between the hugging-dimer and the alternative detached-dimer, this ion is proposed to favor formation ofthe hugging-dimer and, hence, the octamer. Fig. 5B illustrates that removal of magnesium from pea PBGS disfavors the largest form in favor of a smaller form, where the mobility of the two forms is consistent with that of octamer and hexamer. In the model, the hexamer is a putative storage form ofthe PBGS protein because it is less active at physiologic pH and is characterized by a Km value that is well above the physiological concentration of ALA. By contrast, the octamer is active at physiological pH and has a Km value that is in the proper range of ALA concentrations during active tetrapyrrole biosynthesis. Together, these studies support the notion that there is a role for PBGS in the complex control of chlorophyll biosynthesis (26-28). We note that one documented occurrence during the greening of plants is a dramatic increase in the magnesium concentration in the chloroplast
(29). One can imagine that an inactive hexameric storage form allows rapid activation of PBGS as part of a cascade of biochemical changes that accompanies the greening process. It is interesting to note that several gel filtration studies on the quaternary structure of plant and algae PBGS concluded that the oligomer was a hexamer (30 and the references cited therein). Literature support for the existence of interconvertible quaternary forms of PBGS separable by anion exchange chromatography can be found in an early report on PBGS from Chlorella regular is (31). HEXAMERIC HUMAN PBGS REVEALS A NOVEL STRUCTURAL PARADIGM FOR ALLOSTERIC REGULATION OF PROTEIN FUNCTION AND IS THE FIRST EXAMPLE OF A PROTEIN THAT CAN EXIST AS MORPHEINS : Characterization of the human PBGS variant F12L reveals that this point mutation causes a dramatic change in the structure and function of PBGS. This mutation can serve as a precedent for a single amino acid change resulting in significant changes in protein behavior during evolution. The F 12L mutation destabilizes the PBGS octamer and leads to formation of hexamers. The structural transition between octamer and hexamer must proceed through an unprecedented equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS has a binding site in the octamer, but not in the hexamer. Native gel data indicate that removal of the allosteric magnesium favors formation of the hexamer over the octamer. The octamer-hexamer transition defines a novel mechanism for metal ion-dependent allosteric regulation of protein function. This invention describes inhibition of protein function through stabilization of the inactive moφhein of PBGS and/or any other protein that might be regulated by the interconversion of moφheins. In order to decipher molecules that will selectively bind to and stabilize the hexameric form of PBGS, the inventor is taking the following approach. Only those PBGS in QSE are currently being considered as targets because these PBGS have been shown to be active as octamers but they exhibit the protein concentration dependent specific activity phenomenon. The target molecule is one that will selectively bind to the "arm pit" of the hexamer as illustrated by the balls in Fig. 10. The inventor is taking an "in silico" approach of searching molecular libraries for molecules that will bind to the hexameric form of PBGS from the target organisms.
HOMOLOGY MODEL BUILDING FOR TARGET HEXAMERIC PBGS - The only existing crystal structure on which the inventor bases a model of target hexameric PBGS is that of human PBGS clinical variant F12L, PDB code 1PV8 (Breinig et al.
(2003) Nat. Struct. Biol 10, 757-763) Unfortunately, the crystal structure of F12L shows significant disorder, which limits its use as the sole foundation for homology model building. However, comparison of human PBGS octameric and hexameric structures (PDB codes 1E51 and 1PV8) show near identity for the -300 amino acids that comprise a TIM-like alpha, beta- barrel domain. For human PBGS, the difference between octamer and hexamer lies in the structure ofthe 24 amino terminal amino acids and various regions that are more disordered in the hexamer (see Breinig et al.). Hence, one can use a higher quality crystal structure of a PBGS octamer for homology model building the alpha, beta-barrel domain of target PBGS. The chosen structure is PDB code 1GZG ( Frere, F., Schubert, W. D., Stauffer, F., Frankenberg, N., Neier, R, Jahn, D., and Heinz, D. W. (2002) JMol Biol 320, 237-247) Reference (20), which is a highly ordered, high resolution crystal structures of Pseudomonas aeruginosa PBGS, itself a target for inhibitors that would "trap" the PBGS hexamer. A hexameric form of P. aeruginosa PBGS was built using various capacities of Swiss-PDB Viewer (www.expasy.ch spdbv/mainpage.html) and other programs. To build the P. aeruginosa PBGS hexamer, the N-terminal arms were removed from the structure file for the 1GZG dimer. The resulting alpha, beta-barrel domains (residues 32-335) were successively overlaid upon the three dimers of hexameric 1PV8 to create a hexameric assembly ofP. aeruginosa PBGS alpha, beta - barrels. There is no significant sequence identity between the N-terminal arms of human and P. aeruginosa PBGS, but there is a conserved alpha-helix in the structure ofthe N-terminal arm. Hence, a structure alignment of octameric forms of human PBGS and P. aeruginosa PBGS was used to determine the proper sequence alignment for this alpha-helical segment. This information was used to spatially position the amino acids 22-29 of P. aeruginosa PBGS in the hexamer. The program Loopy ( Xiang, Z., Soto, C. S., and Honig, B. (2002) Proc NatlAcadSci USA 99, 7432-7437) was used to model amino acids 29-32, so as to connect the N-terminal alpha-helix to the alpha, beta-barrel domain of each subunit. Finally, the remaining N-terminal amino acids, which are present in file 1PV8, were built onto the P. aeruginosa PBGS structure using phi, psi, and omega angle information for the corresponding amino acids of hexameric human PBGS. Due to disorder in some ofthe N-terminus ofthe human PBGS hexamer (1PV8), the hexamer model for P. aeruginosa PBGS is missing residues 1-9 of subunits A, C, and E as well as residues 1-11 of subunits B, D, and F. The hexameric P. aeruginosa PBGS was the foundation structure for building a model of hexameric pea PBGS using well established published methods as we have done before (Kundrat, L., Martins, J., Stith, L., Dunbrack, R. L., Jr., and Jaffe, E. K. (2003) J Biol Chem 278, 31325-31330) In searching for molecules that will preferentially bind to hexameric PBGS, the following was discovered. Analysis of the hexamer of PBGS shows that the putative
"inhibitor" binding site (also referred to as the arm-pit) contains elements ofthe three subunits A, B, and E. Subunits A and B comprise the already defined "detached dimer", where we typically depict the bottom subunit (subunit A, Fig. 15) such that the reader is looking directly into the active site in the center ofthe alpha, beta-barrel . Subunit B shares a barrel-to-barrel interface with subunit A. Subunit E shares a mutual interaction with subunit B wherein the N- terminal arm of one subunit is nested into the base ofthe alpha-beta-barrel ofthe other subunit. Fig. 15 shows the docked inhibitor, rosmarinic acid, described below. In this docking result, rosmarinic acid has direct interactions with all three subunits shown in the figure. A variety of "Small Molecule" molecular libraries was used, which have been assembled by our collaborator George Markham, and the docking process uses the commercial docking program Glide in an attempt to discover molecules that will trap PBGS in the hexameric form. Working on the presumption that Mother Nature has used the method of moφhein trapping, initial library screening focused on metabolites and natural products. To date, out of a molecular library of- 1 ,000,000 molecules, -30,000 have been screened for molecules that will bind to the "arm pit" ofthe hexameric model of pea PBGS. To date, the best results are with the natural product rosmarinic acid. EXPERIMENTAL DATA WITH ROSMARINIC ACID Inhibition data with rosmarinic acid (Benzenepropanoic acid, D-[[(2E)-3-(3,4- dihydroxyphenyl)-l-oxo-2-propenyl]oxy]-3,4-dihydroxy-, (DR)- (9CI)) is consistent with a slow-tight binding inhibition model wherein rosmarinic acid binds preferentially to the quaternary forms of pea PBGS that are smaller than the octamer. Fig. 16A, open symbols, illustrates the protein concentration dependence of the specific activity of pea PBGS, which shows half maximal activity at 3.5 μg/ml PBGS. This means that at 3.5 μg/ml, under assay conditions, the equilibrium of quaternary isoforms (moφheins) contains about 50% octamer and about 50%) smaller less active isoforms (e.g. hexamers). If an inhibitor acted through preferential binding to these smaller forms, one would expect a more profound inhibition under conditions where the moφhein equilibrium contains these smaller forms. In other words, the inhibitor would be expected to shift the protein concentration dependence to a higher protein concentration, which is shown for rosmarinic acid in FIG 16A (see below). Figs. 16B and 16C show experiments that were done to determine how best to demonstrate this shift in protein concentration dependence. Fig. 16B shows a dose response curve for pea PBGS, which indicates that the IC50 for rosmarinic acid is -63 μM, when the inhibitor is given 30 minutes to act on the protein prior to the addition of substrate. Not shown is the dependence of the inhibition on the preincubation time, where inhibition by any one concentration of rosmarinic acid increases with increasing preincubation time, showing that rosmarinic acid acts as a slow- binding inhibitor. Fig. 16C shows that once inhibition has taken place, the protein does not recover within a 30 minute assay time. The data obtained in Figs. 16A, 16B, and 16C, were used to choose the appropriate conditions necessary to demonstrate the effect of rosmarinic acid on the protein concentration dependence of pea PBGS, as follows. The closed circles of Fig.
16A show the protein concentration dependence ofthe specific activity of pea PBGS following a 30 minute treatment with 30 μM rosmarinic acid, which results in half maximal activity at 13.5 μg/ml PBGS. Thus, following this treatment with rosmarinic acid, the equilibrium of quaternary forms has shifted from 3.5 μM to 13.5 μM; under these conditions and it takes 13.5 μg/ml PBGS to obtain an equilibrium with 50% octamer. This is consistent with the inteφretation that rosmarinic acid stabilizes the smaller, less active forms of PBGS, as illustrated schematically in by the balls in Fig. 10. Fig. 17 supports this conclusion with native gel electrophoresis data. Lanes 2 shows that pea PBGS will separate into at least two quaternary forms. The mobility on the gel is consistent with pea PBGS existing as an equilibrium of octamer and hexamer (see also FIG 5B). Lanes 1 and 3 show that the equilibrium is shifted to smaller foπns following treatment with rosmarinic acid. Lane 1 shows the effects of a 30 minute incubation of 250 μM rosmarinic acid on pea PBGS at 142 μg/ml and lane 3 shows the effect ofthe addition of 10 mM substrate on this quaternary structure equilibrium. Based on our modeling results, the interactions of this biphenyl compound with the "arm pit" ofthe pea PBGS hexamer are predominantly through hydrogen bonds between the protein subunits A, B, and E and the polar moieties of the rosmarinic acid. The protein contains additional hydrogen bonding potential within 4.0 angstroms ofthe rosmarinic acid. Hence, a derivative ofthe rosmarinic acid can be made to have an improved binding by adding additional hydrogen bonding potential to the rosmarinic acid molecule. For instance, one could add a hydroxyl group at the 5 position of either phenyl moiety and improve hydrogen bonding to the protein. Additional hydrophobic interactions with the protein could be obtained by substituting a phenyl or benzyl group at the 2 position ofthe propanoic acid portion ofthe molecule. While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. References 1. Battersby, A.R. Tetrapyrroles: the pigments of life. Not. Prod. Rep. 17, 507-526 (2000).
2. Jaffe, E.K. The poφhobilinogen synthase family of metalloenzymes. Acta Crystallogr. D Biol. Crystallogr. 56, 115-128 (2000). 3. Akagi, R., Yasui, Y., Haφer, P. & Sassa, S. A novel mutation of delta-aminolaevulinate dehydratase in a healthy child with 12% erythrocyte enzyme activity. Br. J. Haematol. 106, 931-937 (1999).
4. Schulze, A., Frommhold, D., Hoffman, G.F. & Mayatepek, E. Spectrophotometric microassay for delta-aminolevlinate dehydratase in dried-blood spots as confirmation for hereditary tyrosinemia type I. Clin. Chem. 47, 1424-1429 (2001).
5. Maruno, M. et al. Highly heterogeneous nature of delta-aminolevulinate dehydratase (ALAD) deficiencies in ALAD poφhyria. Blood 97, 2972-2978 (2001).
6. Jaffe, E.K., Martins, J., Li, J., Kervinen, J. & Dunbrack, R.L., Jr. The molecular mechanism of lead inhibition of human poφhobilinogen synthase. J. Biol. Chem. 276, 1531-1537 (2001).
7. Jaffe, E.K. et al. An artificial gene for human poφhobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning. J. Biol. Chem. 275, 2619-2626 (2000).
8. Jaffe, E.K. et al. Characterization ofthe role ofthe stimulatory magnesium of Escherichia coli poφhobilinogen synthase. Biochemistry 34, 244-251 (1995).
9. Petrovich, R.M., Litwin, S. & Jaffe, E.K. Bradyrhizobium japonicum poφhobilinogen synthase uses two Mg(II) and monovalent cations. J. Biol. Chem. 271, 8692-8699 (1996).
10. Frankenberg, Ν., Jahn, D. & Jaffe, E.K. Pseudomonas aeruginosa contains a novel type N poφhobilinogen synthase with no required catalytic metal ions. Biochemistry 38, 13976- 13982 (1999).
11. Erskine, P.T. et al. X-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase. Nat. Struct. Biol. 4, 1025-1031 (1997).
12. Erskine, P.T. et al. X-ray structure of 5 -aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution. Biochemistry 38, 4266-4276 (1999).
13. Erskine, P.T. et al. The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid. Protein Sci. 8, 1250-1256 (1999).
14. Frankenberg, Ν. et al. High resolution crystal structure of a Mg2+-dependent poφhobilinogen synthase. J. Mol. Biol. 289, 591-602 (1999). 15. Erskine, P.T. et al. MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites. Acta Crystallogr. D Biol. Crystallogr. 56, 421-430 (2000).
16. Kervinen, J. et al. Mechanistic basis for suicide inactivation of poφhobilinogen synthase by 4,7-dioxosebacic acid, an inhibitor that shows dramatic species selectivity. Biochemistry 40, 8227-8236 (2001).
17. Erskine, P.T. et al. The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors. FEBS Lett. 503, 196-200 (2001). 18. Erskine, P.T. et al. The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors. J. Mol. Biol. 312, 133-141 (2001).
19. Jaffe, E.K. et al. Species-specific inhibition of poφhobilinogen synthase by 4-oxosebacic acid. J. Biol. Chem. 277, 19792-19799 (2002).
20. Frere, F. et al. Structure of poφhobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism. J. Mol. Biol. 320, 327-247 (2002).
21. Murzin, A.G., Brenner, S.E., Hubbard, T. & Chothia, C. SCOP: a structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247, 536-540 (1995). 22. Kervinen, J. et al. Poφhobilinogen synthase from pea: expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions. Biochemistry 39, 9018-9029 (2000).
23. Segel, I.H. Enzyme Kinetics. 64-71 (John Wiley & Sons, Inc., 1975).
24. Jaffe, E.K. An unusual phylogenetic variation in the metal ion binding sites of poφhobilinogen synthase. Chem. Biol. 10, 25-34 (2003).
25. Frankenberg, N . , Heinz, D . W. & Jahn, D . Production, purification, and characterization of a Mg2+-responsive poφhobilinogen synthase from Psuedomonas aeruginosa. Biochemistry 38, 13968-13975 (1999).
26. Schneider, H.A. Enzymic capacities for chlorophyll biosynthesis. Activation and de novo synthesis of enzymes. Z Naturforsch 31, 55-63 (1976).
27. Papenbrock, J., Mock, H.P., Tanaka, R., Kruse, E. & Grimm, B. Role of magnesium chelatase activity in the early steps ofthe tetrapyrrole biosynthetic pathway. Plant Physiol. 122, 1161-1169 (2000).
28. Papenbrock, J. & Grimm, B. Regulatory network of tetrapyrrole biosynthesis—studies of infracellular signalling involved in metabolic and developmental control of plastids. Planta 213, 667-681 (2001).
29. Walker, D.A. Curr. Top. Cell Regul. 11, 203-241 (1976).
30. Stolz, M. & Dornemann, D. Purification, metal cofactor, N-terminal sequence and subunit composition of a 5 -aminolevulinic acid dehydratase from the unicellular green alga Scenedesmus obliquus, mutant C-2A'. Ewr. J. Biochem. 236, 600-608 (1996).
31. Tamai, H., Shioi, Y. & Sasa, T. Plant Cell Physiol. 20, 435-444 (1979).
32. Laue, T., Shaw, B.D., Ridgeway, T.M. & Pelletier, S.L. in Analytical Ultracentrifugation in Biochemistry and Polymer Science (eds. Harding, S.Ε., Rowe, A. & Horton, J.C.) 90- 125 (The Royal Society of Chemistry, Cambridge, UK, 1992).
33. Jaffe et al., "Dissection of the early steps in the poφhobilinogen synthase-catalyzed reaction: requirements for Schiff s base formation." J. Biol. Chem.267, 9348-9353. 1986.
34. Edwards et al. (1990). Proc Natl Acad Sci USA 87 (9): p3459-3463.
35. Lloyd et al. (1991). Mol. Gen. Genet. 225 (2):209-216.
36. Stockhaus et al. (1989). EMBO Journal 8(9):2445-2451. 37. Leyva et al. (1995) Plant Physiology 108(l):39-46.
38. Campbell et al. (1994) Canadian Journal of Forest Research 24 (8): 1689-1693.
39. Fejes et al. (1990). Plant Mol Biol 15 (6): p921-932.
40. Luebberstedt et al. (1994) Plant Physiology 104 (3):997-1006.
41. Luan et al. (1992). Plant Cell 4 (8):971-981. 42. Matsuoka et al. (1993). Proc. Natl. Acad. Sci. U.S.A. 90(20):9586-9590.
43. Cerdan et al. (1997) Plant Molecular Biology 33 (2): p245-255.
44. Truernit et al. (1995) Planta 196 (3):564-570.
45. Oelmueller et al. (1992). Res. Photosynth. Proc. Int. Congr. Photosynth., 9th Volume 3: p219-24. Editor(s): Murata, Norio. Publisher: Kluwer, Dordrecht, Neth.
46. Kretsch et al. (1995) Plant Journal 7 (5): p715-729.
47. - Bevan et al. (1986) Nucleic Acids Res. 14 (11):4625-4638.
48. Jefferson et al. (1990) Plant Mol. Biol. 14: 995-1006. 49. Muller et al (1990) Mol. Gen. Genet. 224:136-146.
50. Salanoubat and Belliard (1987) Gene 60:47-56.
51. Salanoubat and Belliard (1989) Gene 84:181-185.
52. Hannapel (1990) Plant Physiol. 94: 919-925.
53 Rohde et al. (1990) J. Genet. & Breed, 44:311-315. 54. Rocha-Sosa et al. (1989) EMBO J. 8 (l):23-29.
55. Mignery et al (1988) Gene 62:27-44.
56. Tierney et al. (1987) Planta 172:356-363.
57. Pedersen et al. (1982) Cell 29:1015-1026. 58. Zheng et al. (1993) Plant J. 4: 3357-3366.
59. Russell and Fromm (1997) Transgenic Research 6 (2):157-168.
60. Samac et al. (1990) Plant Physiol. 93:907-914.
61. Suzuki et al. (1994) Plant Mol. Biol. 25(3):507-516. 6.2.1 Daniell et al. (1998) Nature Biotechnology 16:345-348.
63. Daniell et al., "Containment of herbicide resistance through genetic engineering ofthe chloroplast genome," Nature Biotechnology, 16: 345-348 (1998). 64. Potrykus et al. (1985), Mol. Gen. Genet. 199:183-188.
65. Potrykus, I., et al., "Direct Gene Transfer to Cells of a Graminaceous Monocot", Mol. Gen. Genet, 199, pp. 183-188, (1985). 66. Hinchee et al., Bio/Technoloqy 6:915-922 (1988).
67. Hinchee, M.A., et al., "Production of Transgenic Soybean Plants Using Agrobacterium- Mediated DNA Transfer", Bio/Technology, 6, pp. 915-922, (Aug. 1988) 68. Stalker et al. (1988) J. Biol. Chem. 263:6310-6314.
69. Stalker, D.M., et al., "Herbicide Resistance in Transgenic Plants Expressing a Bacterial Detoxification Gene", Science, 242, pp. 419-422, (Oct. 21, 1988). 70. EP 0154204 Sep., 1985.
71. Thillet et al. (1988) J. Biol. Chem. 263:12500-12508.
72. Thillet, J., et al., "Site-directed Mutagenesis of Mouse Dihydrofolate Reductase", The Journal of Biological Chemistry, 263 (25), pp. 12500-12508, (Sep. 1988).
73. Herrera-Estrella et al. (1983) Nature 303:209.
74. Bevan (1984) Nucleic Acids Res. 12 (22): 8711-8721.
75. Klee et al. (1985) Bio-Technology 3(7): 637-642.
76. Daniell et al. (1998) Nature Biotechnology 16:345-348. 77. Daniell et al., "Containment of herbicide resistance through genetic engineering ofthe chloroplast genome," Nature Biotechnology, 16: 345-348 (1998).
78. Pedersen et al. (1982) Cell 29:1015-1026. 79. Fraley et al. (1983) Proc Natl Acad Sci USA 80: 4803-4807
80. Rogers, S.G., et al., "Chapter 15: Improved Vectors for Plant Transformation: Expression Cassette Vectors and New Selectable Markers", In: Methods in Enzymology, V.153: Recombinant DNA, Part D, Academic Press, Inc.: Ray Wu, et al., (Eds.), pp. 253-277 (1987).
81. Rogers et al. (1987) Improved vectors for plant transformation: expression cassette vectors and new selectable markers. In Methods in Enzymology. Edited by R. Wu and L. Grossman. p253-277. San Diego: Academic Press.
82. Schmidhauser and Helinski. (1985) J. Bacteriol. 164-155.
83. Horsch and Klee. (1986) Proc. Natl. Acad. Sci. U.S.A. 83:4428-4432.
84. Ammirato et al. ( 1984) Handbook of Plant Cell Culture-Crop Species. Macmillan Publ. Co. 85. Fromm, M., (1990) UCLA Symposium on Molecular Strategies for Crop Improvement, Apr. 16-22, 1990. Keystone, Colo.
86. Vasil et al. (1990) Bio/Technology 8:429-434. 87. Vasil et al. (1992) Bio/Technology 10:667-674.
88. Hayashimoto et al. (1990) Plant Physiol. 93:857-863. 89. Datta et al. (1990) Bio-technology 8:736-740. 90. Breinig et al. (2003) Nat. Struct. Biol 10, 757-763) 91. ( Xiang, Z., Soto, C. S., and Honig, B. (2002) Proc Natl Acad Sci USA 99, 7432- 7437) 92. (Kundrat, L., Martins, J., Stith, L., Dunbrack, R. L., Jr., and Jaffe, E. K. (2003) J Biol Chem 278, 31325-31330) 93. PCT Publication WO 84/02913 94. PCT Publication WO93/19189 95. PCT Publication WO 92/04449

Claims

CLAIMS WHAT IS CLAIMED IS: 1. A composition comprising an agent adapted to affect a multimeric protein by binding to a binding site of said multimeric protein and thereby affecting an equilibrium of units, wherein said multimeric protein comprises an assembly having a plurality of said units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that in said multimeric protein ( 1 ) a structure of each of said units determines a structure of said different quaternary isoforms, (2) said units are in the equilibrium and (3) the structure of said different quaternary isoforms influences a function ofthe multimeric protein.
2. The composition of claim 1, wherein affecting said multimeric protein comprises affecting a formation of a quaternary isoform.
3. The composition of claim 1, wherein affecting said multimeric protein comprises affecting the function of said multimeric protein.
4. The composition of claim 3, wherein the function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating.
5. The composition of claim 4, wherein the agent is bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity.
6. The composition of claim 1, wherein each of said units is a member selected from the group consisting of a monomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer.
7. The composition of claim 1, wherein said multimeric protein is a member selected from the group consisting of poφhobilinogen synthase and a Class la ribonucleotide reductase.
8. The composition of claim 7, wherein said multimeric protein is poφhobilinogen synthase comprising eight poφhobilinogen synthase monomers.
9. The composition of claim 8, wherein the agent is an inhibitor bound to the quaternary isoform having the lesser activity and wherein the quaternary isoform contains less than eight poφhobilinogen synthase monomers.
10. The composition of claim 7, wherein said multimeric protein is the Class la ribonucleotide reductase and the agent inhibits the Class la ribonucleotide reductase through selective binding to the binding site that is unique to the quaternary isoform having the lesser activity.
11. A composition comprising an inhibitor adapted to inhibit formation of an active form of a multimeric poφhobilinogen synthase having a first number of monomers by binding to a less active form of the multimeric poφhobilinogen synthase having a second number of monomers, wherein the first number of monomers is higher than the second number of monomers.
12. The composition of claim 11 , wherein the multimeric poφhobilinogen synthase is derived from bacteria, archaea, or eucarya, provided that the octameric poφhobilinogen synthase contains an allosteric magnesium binding site.
13. The composition of claim 12, wherein the multimeric poφhobilinogen synthase contains a catalytic zinc binding site.
14. The composition of claim 11 , wherein the multimeric poφhobilinogen synthase does not contain the allosteric magnesium binding site and the catalytic zinc binding site.
15. The composition of claim 11 , wherein said less active form is a hexamer.
16. The composition of claim 11 , wherein said less active form is a dimer.
17. The composition of claim 11, wherein the inhibitor replaces a metal ion and thereby binds at a metal ion binding site.
18. The composition of claim 17, wherein the metal ion is zinc and/or magnesium.
19. The composition of claim 11 , wherein the inhibitor binds at an active site.
20. The composition of claim 11 , wherein the inhibitor is not a metal cation.
21. The composition of claim 11, wherein the inhibitor is adapted to inhibit formation ofthe active form ofthe multimeric poφhobilinogen synthase, said active form is an octomeric poφhobilinogen synthase by binding to a hug-disabling domain of the less active form ofthe multimeric poφhobilinogen synthase containing less than eight monomers.
22. The composition of claim 11, wherein the inhibitor is adapted to inhibit formation of the active form of the multimeric poφhobilinogen synthase by binding at a site other than an active site and/or metal ion binding site.
23. The composition of claim 11, wherein the inhibitor is adapted to inhibit formation ofthe active form ofthe multimeric poφhobilinogen synthase by a mechanism other than removing a metal ion.
24. The composition of claim 11 , further comprising a delivery medium, said delivery medium is a member selected from the group consisting of a pharmaceutically- acceptable medium, an orally-acceptable carrier, an antibacterial medium, and a herbicidally- effective medium.
25. The composition of claim 24, wherein the composition is effective to inhibit or prevent formation ofthe active form ofthe multimeric poφhobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric poφhobilinogen synthase contains an allosteric magnesium binding site.
26. The composition of claim 25, effective to cure or prevent a disease caused by contacting bacteria, archaea, and/or eucarya.
27. The composition of claim 25, wherein the composition is at least one of a drug, a toothpaste, a soap, a desinfectant, an anti-biofilm composition, and a herbicide.
28. The composition of claim 24, wherein the composition is effective to inhibit or prevent formation ofthe active form ofthe multimeric poφhobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form ofthe multimeric poφhobilinogen synthase does not contain the allosteric magnesium binding site and the catalytic zinc.
29. The composition of claim 28, effective to cure or prevent a disease or caused by contacting bacteria, archaea, and/or eucarya.
30. The composition of claim 28, wherein the composition is at least one of a drug, a toothpaste, a soap, and a disinfectant.
31. A herbicide resistant plant adapted to be transgenic for a multimeric poφhobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.
32. The herbicide resistant plant of claim 31, wherein the multimeric poφhobilinogen synthase is derived from a human.
33. The herbicide resistant plant of claim 31, wherein the multimeric poφhobilinogen synthase contains no allosteric magnesium binding site.
34. A composition comprising an inhibitor adapted to bind to a multimeric poφhobilinogen synthase that does not require zinc for catalytic function.
35. A method of affecting a multimeric protein, the method comprising: providing said multimeric protein comprising an assembly having a plurality of units, wherein each of said units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1 ) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function of said multimeric protein; providing the composition of claim 1 comprising the agent, wherein the agent is adapted to affect the equilibrium by binding to a binding site on the assembly; and contacting the assembly with the agent, wherein the agent affects the equilibrium by binding to the binding site and thereby affecting said multimeric protein.
36. The method of claim 35, wherein affecting said multimeric protein comprises affecting a formation of a quaternary isoform.
37. The method of claim 35, wherein affecting said multimeric protein comprises affecting a function of said multimeric protein.
38. The method of claim 35, wherein the unit is a member selected from the group consisting of a momomer, a dimer, a trimer, a tetramer, a hexamer, and an octamer.
39. The method of claim 35, wherein the agent is adapted to affect a function of said multimeric protein.
40. The method of claim 39, wherein the function of said multimeric protein is an activity and wherein affecting is at least one of inhibiting or activating.
41. The method of claim 40, wherein the agent is bound to at least one of a quaternary isoform having a lesser activity or a quaternary isoform having a greater activity.
42. The method of claim 41 , wherein the agent is bound to the quaternary isoform having a greater activity.
43. The method of claim 35 , wherein said multimeric protein is a member selected from the group consisting of poφhobilinogen synthase and a Class la ribonucleotide reductase.
44. The method of claim 43, wherein said multimeric protein is poφhobilinogen synthase comprising eight poφhobilinogen synthase monomers.
45. The method of claim 43, wherein said multimeric protein is the Class la ribonucleotide reductase and the agent inhibits the Class la ribonucleotide reductase through selective binding to the binding site that is unique to the quaternary isoform having the lesser activity.
46. A method of modulating a physiological activity in a cell, a tissue or an organism, the method comprising: providing the cell, the tissue or the organism, wherein the cell, the tissue or the organism comprise a multimeric protein comprising an assembly having a plurality of units, wherein each ofthe units comprises a first complementary surface and a second complementary surface and wherein the first complementary surface of one unit is associated with the second complementary surface of another unit, provided that the assembly is at least one of different quaternary isoforms on a condition that (1) a structure of said units determines a structure of said different quaternary isoforms, (2) said units are in an equilibrium and (3) the structure of said different quaternary isoforms influences a function ofthe multimeric protein; and providing the composition of claim 1 comprising the agent to the cell, the tissue or the organism, wherein the agent is adapted to affect the equilibrium by binding to the binding site on the unit and thereby affecting the formation of a quaternary isoform and thereby modulating the physiological activity.
47. A method of inhibiting a multimeric poφhobilinogen synthase from forming an active form, the method comprising: applying the composition of claim 11 to the multimeric poφhobilinogen synthase; associating the composition with the less active form; inhibiting the less active form from assembling into the active form and thereby inhibiting the multimeric poφhobilinogen synthase from forming the active form.
48. A method for manipulating growth or development of a plant comprising applying the composition of claim 27 to the plant, wherein the plant is herbicide resistant and is adapted to be transgenic for a multimeric poφhobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.
49. The method of claim 48, wherein the multimeric poφhobilinogen synthase contains no allosteric magnesium binding site.
50. A method of making an antibacterial surface, the method comprising: providing the composition of claim 27; providing a surface-forming matrix; combining the composition with the surface-forming matrix and thereby making the antibacterial surface.
51. The method of claim 50, wherein the antibacterial surface is adapted to prevent or inhibit a formation of a biofilm.
52. The composition of claim 11, wherein the inhibitor is rosmarinic acid or a derivative thereof.
PCT/US2004/021722 2003-07-07 2004-07-07 Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides WO2005007817A2 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
JP2006518868A JP2007529995A (en) 2003-07-07 2004-07-07 Hexameric porphobilinogen synthase as a target for developing antibiotics and herbicides
EP04786044A EP1648429A4 (en) 2003-07-07 2004-07-07 Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
AU2004257208A AU2004257208B2 (en) 2003-07-07 2004-07-07 Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
CA002531692A CA2531692A1 (en) 2003-07-07 2004-07-07 Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
US11/327,762 US20060162014A1 (en) 2003-07-07 2006-01-06 Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
IL173016A IL173016A (en) 2003-07-07 2006-01-08 Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
US12/106,498 US7863029B2 (en) 2004-06-04 2008-04-21 Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
US12/142,435 US8153410B2 (en) 2003-07-07 2008-06-19 Alternate morpheein forms of allosteric proteins as a target for the development of bioactive molecules
US12/942,320 US20110207816A1 (en) 2003-07-07 2010-11-09 Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US48525303P 2003-07-07 2003-07-07
US60/485,253 2003-07-07
US57731204P 2004-06-04 2004-06-04
US60/577,312 2004-06-04

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US11/327,762 Continuation-In-Part US20060162014A1 (en) 2003-07-07 2006-01-06 Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules

Publications (3)

Publication Number Publication Date
WO2005007817A2 true WO2005007817A2 (en) 2005-01-27
WO2005007817A3 WO2005007817A3 (en) 2005-11-03
WO2005007817A9 WO2005007817A9 (en) 2006-04-06

Family

ID=34083333

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/021722 WO2005007817A2 (en) 2003-07-07 2004-07-07 Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides

Country Status (6)

Country Link
EP (1) EP1648429A4 (en)
JP (1) JP2007529995A (en)
AU (1) AU2004257208B2 (en)
CA (1) CA2531692A1 (en)
IL (1) IL173016A (en)
WO (1) WO2005007817A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006138466A3 (en) * 2005-06-15 2008-02-28 Fox Chase Cancer Ct Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
US8153410B2 (en) 2003-07-07 2012-04-10 Fox Chase Cancer Center Alternate morpheein forms of allosteric proteins as a target for the development of bioactive molecules

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69930619T2 (en) * 1998-05-16 2006-12-28 Mogam Biotechnology Research Institute, Yongin USE OF ROSMARIC ACID AND ITS DERIVATIVES AS IMMUNOSUPPRESSOR OR AS INHIBITOR OF SH-2-MEDIATED PROCESSES
JP4112730B2 (en) * 1999-02-22 2008-07-02 オリザ油化株式会社 Antibacterial agent for oral cavity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP1648429A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8153410B2 (en) 2003-07-07 2012-04-10 Fox Chase Cancer Center Alternate morpheein forms of allosteric proteins as a target for the development of bioactive molecules
US7863029B2 (en) 2004-06-04 2011-01-04 Fox Chase Cancer Center Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
WO2006138466A3 (en) * 2005-06-15 2008-02-28 Fox Chase Cancer Ct Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
EP1898692A2 (en) * 2005-06-15 2008-03-19 Fox Chase Cancer Center Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
EP1898692A4 (en) * 2005-06-15 2008-08-13 Fox Chase Cancer Ct Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
JP2008546389A (en) * 2005-06-15 2008-12-25 フォックス チェイス キャンサー センター Alternative morphine of allosteric protein as a target for developing bioactive molecules

Also Published As

Publication number Publication date
AU2004257208A1 (en) 2005-01-27
IL173016A (en) 2012-03-29
CA2531692A1 (en) 2005-01-27
JP2007529995A (en) 2007-11-01
WO2005007817A3 (en) 2005-11-03
AU2004257208B2 (en) 2010-02-25
EP1648429A2 (en) 2006-04-26
EP1648429A4 (en) 2009-09-02
IL173016A0 (en) 2006-06-11

Similar Documents

Publication Publication Date Title
US8153410B2 (en) Alternate morpheein forms of allosteric proteins as a target for the development of bioactive molecules
JP6995627B2 (en) Compositions for treating pathological calcification conditions and methods of using them
EP0668933B1 (en) Use of methioninase as an antitumor agent in anti-methionine chemotherapy
US6613553B1 (en) Enoyl reductases and methods of use thereof
CA2341412A1 (en) Rationally designed heparinases derived from heparinase i and ii
JP6665281B2 (en) Human fibroblast growth factor-2 mutant with increased stability and use thereof
US20110207816A1 (en) Alternate morpheeins of allosteric proteins as a target for the development of bioactive molecules
RU2652605C2 (en) Blocking of inflammatory proteases by theta-defensins
AU2004257208B2 (en) Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
WO2005007817A9 (en) Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
WO2014059313A1 (en) Thermally stable enzymes, compositions thereof and methods of using same
TWI754828B (en) Powdery composition of ogg1 and uses thereof
US6638744B2 (en) Canine cox-2 nucleic acid molecules and uses thereof
EP1069911A1 (en) A novel nitroreductase and therapeutic uses therefor
CN100390286C (en) Novel enzyme gene and its expression product
WO2020152532A1 (en) Method of ameliorating a pro-inflammatory immunophenotype in farber disease subjects by repeated administration of a recombinant human acid ceramidase
CN113939586A (en) Methods of treating beta-thalassemia
JP3355368B2 (en) Plasmid vector containing cDNA encoding plant-derived epoxide hydrolase and transformant
TWI361041B (en) Gene, protein and method for improving aroma production in plants
CN1835739A (en) Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides
HUT54208A (en) Process for producing new serine protease inhibitor proteins, pharmaceutical compositions comprising same, dna sequences coding for such proteins and expression vectors containing such dna sequences
JPH10505747A (en) Human inositol monophosphatase H1
WO2010029378A2 (en) Anti-protozoa compounds
WO2023028586A1 (en) Use of taxifolin for treating systemic lupus erythematosus
Parker Production and purification of imidazole glycerol phosphate dehydratase from Escherichia coli and Cryptococcus neoformans: Comparative kinetic and metal binding studies

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200480023403.2

Country of ref document: CN

AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2531692

Country of ref document: CA

Ref document number: 11327762

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2006518868

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2004257208

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2004786044

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2004257208

Country of ref document: AU

Date of ref document: 20040707

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 2004257208

Country of ref document: AU

COP Corrected version of pamphlet

Free format text: PAGES 4, 9-14, 37, 39, AND 50, DESCRIPTION, REPLACED BY NEW PAGES 4, 9-14, 37, 39 AND 50; AFTER RECTIFICATION OF OBVIOUS ERRORS AUTHORIZED BY THE INTERNATIONAL SEARCH AUTHORITY

WWP Wipo information: published in national office

Ref document number: 2004786044

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 11327762

Country of ref document: US