CA2531692A1 - Hexameric porphobilinogen synthase as a target for the development of antibiotics and herbicides - Google Patents

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 iri 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 T#-IE INVENTION
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 2 0 common to tetrapyrrole biosynthesis in all organisms.
The enzyme porphobilinogen synthase (PBGS), also known as S-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 oftwo 5-aminolevulinic acid molecules to form 2 5 the tetrapyrrole precursor porphobilinogen.
PBGS was previously understood to be a homooctameric metalloenzyme, which utilizes a variety of divalent and monovalent canons 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 3 0 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 3 5 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 ethylenediaminetetraacetic acid (EDTA).
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 l0 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 of the 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.
2 0 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 2 5 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 sufFcient 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 3 0 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 ofPseudomonas 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 3 5 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 2 0 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 2 5 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 3 0 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 3 5 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 Ia 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 Ia ribonucleotide reductase and the agent inhibits the Class Ia 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 2 0 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 2 5 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, 3 0 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.
3 5 In certain embodiments, the inhibitor is not a metal cation.

In certain embodiments, the inhibitor is adapted to inhibit formation of the active form of the multimeric porphobilinogen synthase, said active form is an octomeric porphobilinogen 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 of the active form of the multimeric porphobilinogen 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 of the active form of the multimeric porphobilinogen synthase by a mechanism other than removing a metal ion.
l0 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 of the active form of the multimeric porphobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric porphobilinogen 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 2 0 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 of the multimeric porphobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the 2 5 multimeric porphobilinogen 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 3 0 porphobilinogen synthase that substantially exist in a multimeric form of a hugging dimer. In certain embodiments, the multimeric porphobilinogen synthase is derived from a human. In certain embodiments, the multimeric porphobilinogen synthase contains no allosteric magnesium binding site.
Further provided is a composition comprising an inhibitor adapted to bind to a 3 5 multimeric porphobilinogen 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 of the 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.
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 of the method, the agent is adapted to affect a function of said multimeric protein.
2 0 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 of the 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 2 5 having a greater activity.
In certain embodiments of the method, said multimeric protein is a member selected from the group consisting of porphobilinogen synthase and a Class Ia ribonucleotide reductase.
In certain embodiments of the method, said multimeric protein is porphobilinogen synthase comprising eight porphobilinogen synthase monomers.
3 0 In certain embodiments of the method, said multimeric protein is the Class Ia ribonucleotide reductase and the agent inhibits the Class Ia 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 3 5 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 of the 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 isofonn and thereby modulating the physiological activity.
Further provided is a method of inhibiting a multimeric porphobilinogen synthase from forming an active form, the method comprising: applying the composition of the invention to the multimeric porphobilinogen 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 porphobilinogen synthase from forming the active form.
Further 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 2 0 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.
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 2 5 prevent formation of the active form of the multimeric porphobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric porphobilinogen 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 3 0 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 3 5 reference numerals designate like elements and wherein:

Fig. 1 A shows the pH rate profile for wild type human PBGS relative to the F

variant.
Fig. 1 B shows the chromatographic separation of wild type human (Wt) PBGS and on a mono-Q column.
Fig. 1 C shows the differential mobility of wild type (Wt) human PBGS and F
12L on 12.5% polyacrylamide native gel electrophoresis.
Fig. 2A 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 (KCl 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 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 5300 purified Pool I (circles) and Pool II (squares) at pH
7 (black) and pH 9 (gray).
2 0 Fig. 4A shows a schematic representation of the crystal structure of E.
coli PBGS, including the location of the allosteric magnesium.
Fig. 4B shows schematic representations of the subunit interfaces of human PBGS
octamer (left) relative to the hexamer (right).
Fig. SA shows a schematic representation of the equilibrium existing between dimeric, 2 5 hexameric and octameric PBGS.
Fig. SB 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 3 0 (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.
3 5 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 l0 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 2 0 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 of the magnesium. R11 derives from the N-terminal arm of the 2 5 neighboring subunit of the hugging dimer.
Fig. 10 shows a schematic representation of an embodiment of the 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.
3 0 Fig. 11 is a two-dimensional schematic representation of the equilibrium between two isoforms of a protein demonstrating that an agent that is capable of affecting a function of the 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 of the equilibrium between two 3 5 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 of the monomer dictates the oligomeric state and the oligomeric state dictates the functional characteristics. In each case, two different conformations of the unit must be utilized in order to equilibrate between the multimers.
Fig. 14 is a two-dimensional schematic representation of the 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. 16A 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 p,g/ml) and the filled circles form a 2 0 curve wherein rosmarinic acid was used (Kd = 13.5 pg /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 2 5 of pea PBGS (Pea PBGS at 10 mg/ml, 30 min preincubation with rosmarinic acid. ICSO = 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 3 0 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 "morpheins" can be modulated by agents (e.g., small molecules) that bind to the unique surface features of one or another of these morpheins and thus shift the equilibrium of quaternary forms.
3 5 According to certain embodiments of the invention, tetrapyrrole biosynthesis can be modulated by modulating the equilibrium among the morpheins of PBGS. According to certain embodiments of the invention, inhibitor molecules can be discovered that will preferentially interact with the unique surface components of the PBGS hexamer and displace the distribution of morpheins 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 of the multimeric protein. The composition of the 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 of the 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 2 0 desired effect.
The unit of the 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 2 5 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 porphobilinogen synthase and a Class Ia 3 0 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 porphobilinogen synthase monomers. Similarly, when the multimeric protein is the Class Ia ribonucleotide reductase, the agent inhibits the Class Ia ribonucleotide reductases through selective binding to the binding site that is unique to a less active quaternary isoform.
3 5 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 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 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 a8(I8 barrel comprised of the C-l0 terminal 300 amino acids, wherein the center of the a8(38 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 of the N-terminal arm.
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 The following concepts and definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.
MORPHEINS CONCEPT:
One dogma of modern biochemistry is that the three dimensional structure of a protein is 2 5 a direct consequence of the 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 morpheins, which are alternate protein quaternary structures that are a physiologically relevant consequence of a conformational change in the monomeric 3 0 unit. In the case of morpheins, 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 morpheins is the porphobilinogen synthase (PBGS) system where the quaternary structure equilibrium forms the structural basis for the phenomenon of allosterism.
3 5 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 of the concept that the multiplicity of the assembly is directed by the shape of the 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 of the dashed lines are adjacent to all of the 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 morphein 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 of the different oligomers might be a result of differences in the molecular surface of the oligomer: For instance, the rounded surface of the trimer in Figs. 11 - 14 would interact with different receptors or binding partners than the oval surface of the dimer or the pointed surface of the tetramer. These molecular surface differences could dictate 2 0 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 of the same multiplicity, and that these two forms are in equilibrium with each other, 2 5 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 of the enzyme porphobilinogen synthase (PBGS) wherein one can 3 0 examine these forms and deduce a rational explanation for the allosteric regulation of the PBGS
of some organisms by magnesium. This discovery is the first concrete example of morpheins 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 morpheins as a regulatory mechanism for protein function and the description of agents that can trap one or 3 5 another of the morphein forms such as to direct protein function. Fig. l l 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 of the protein and thus act to draw the equilibrium toward that state. Thus, these agents, which will perturb the quaternary structure equilibrium of morpheins, can inhibit or activate the protein. In one embodiment of the invention, the agent is an inhibitor, which traps the inactive form and therefore prevents formation of the 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 of the inhibitor is a rosemarinic acid or a derivative thereof. In another embodimentof the invention, the agent is an activator, which traps the active form of the protein.
The term "promoter" or "promoter region" refers to a nucleic acid sequence, usually found upstream (S') to a coding sequence, that controls expression of the 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 2 0 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 2 5 (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is incorporated 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.
3 0 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 3 5 attached.

The term "encoding DNA" refers to chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA that encodes any of the enzymes discussed herein.
The term "genome" as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs of the 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 of the cell. DNAs of the present invention introduced into plant cells can therefore be either chromosomally integrated or organelle-localized.
l0 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 porphobilinogen 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.
2 0 "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.
2 5 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, 3 0 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 3 5 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 purposes 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 purposes 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 "morpheins" is used in this disclosure by the inventor to describe different quaternary isoforms of a protein with different functional characteristics (e.g., different catalytic 2 0 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 morpheins is the allosteric regulator, i.e., an agent (e.g., magnesium).
2 5 Morpheins of a given protein have partial differences in secondary and tertiary structure, and these differences dictate a difference in quaternary structure. In certain aspects, morpheins 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, morpheins are unlike prions in that the oligomer is of finite mulitplicity and the quaternary structure change is 3 0 reversible, non-pathologic, and part of a normal physiologic control process.
Accordingly, in this the invention, a general mechanism for allosteric regulation using morpheins (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 morphein. This mechanism is illustrated schematically 35 in Figs.ll-14.

Fig. 11 is a two dimensional representation of the equilibrium between two forms of a protein (morpheins). 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 of the 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 of the 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 of the multimeric protein, for example, in the case of plant and some bacterial PBGS, magnesium provides stability to these forms of the 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 2 0 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 of the splinter will_pull the equilibrium of quaternary 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
2 5 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. SA).
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 3 0 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 3 5 with signal transduction. What has not been appreciated prior to this invention is that the structures of the 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 morpheins is a Class Ia ribonucleotide reductase. The recent model put forth for allosteric regulation of Class Ia ribonucleatide 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 morpheins. However, ribonucleotide reductase is essential for de novo DNA
biosynthesis, the Class Ia 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 Ia 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 morphein.
The multimetic protein of the 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, Km and Vmax~ activity as a function of substrate concentration fit to MM equation;
morpheins will not fit well to a hyperbolic curve but rather a double hyperbolic curve. (see Nature Strucure Biology) 2 0 Non-limiting example of a function of the 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", and V",~ as a skilled artesian would appreciate. In certain embodiments of the invention, inhibition of a protein function 2 5 through stabilization of a less active morphein.
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 3 0 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 3 5 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 l0 change in the protein structure. See, e.g., Fig. SA. Certain embodiments of the invention relate to the inhibition of the 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 of the 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 2 0 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 2 5 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 of the allosteric magnesium is to induce a structural change between the low activity hexamer and the high activity octamer. The hexamer-octamer transition for Mg 3 0 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 3 5 many PBGS. This zinc functions in the binding and reactivity of the 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 l0 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% of the PBGS sequences contain the determinants for allosteric magnesium binding (i.e., QSW + QSE » QNW + QNE).
2 0 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 2 5 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 3 0 sources of the octameric PGBS, which can be inhibited by the composition of the 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 of the four quadrants of Fig. 6 is based on the 3 5 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 of the 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 anywhere, 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 of the 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 of the 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 of the subunit (i.e., at the "arm-pit"). Theoretically suitable molecules will be empirically tested in vitro by determining 2 0 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 2 5 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 3 0 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 3 5 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 the 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 porphobilinogen synthase from forming an active form, the method comprising: applying the composition of the invention to the multimeric porphobilinogen 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 porphobilinogen 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 2 0 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.
2 5 Using Table I 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 S ecies name Potential Dama a Yersinia enterocolitica Food borne astroenteritis Yersinia estis Pla ue Pseudomonas s rin ae Plant atho en tomatoes Pseudomonas aeruginosa Opportunistic human pathogen of compromised tissues. Notorious for antibiotic resistance Actinobacillus actinom cetemcomitansPeriodontal disease Pasturella multocida ~ Invective agent in animal bite wounds Shewenella utre asciens Oil i eline corrosion, fish s oils a Meth lococcus ca sulatus Uses methane as carbon source Vibrio cholerae Cholera - severe diarrhea X lella astidiosa Pierces disease in lams e. .
ra es Caulobacter crescentus As mmetric cell division Agrobacterium tumefaciens Crown gall on rose and others like apple, ear, each, the , etc.

Sinorhizobium meliloti Nitro en fixin bacteria for alfalfa Brucella melitensis Bacterial disease of domestic animals shee , oats . Malta fever in humans.

Rhodo seudomonas alustris Pu le non-sulfur hototro is bacterium.

Mesorhizobium loti Biotech sub'ect Brad rhizobium 'a onicum Nitro en fixation so beans.

Brucella melitensis biovarsvis Brucellosis - zoonotic disease Ma netos irillum ma netotacticumForms ma netite Rickettsia conorii Mediterranean s otted fever Rickettsia rowazekii E idemic t hus Novos hin obium aromaticivoransFood indust Bordetella bromchse tica Common in cats Bordetella ertussis Whoo in cou h Nitrosomonas euro aea Auxotro is nitri in bacteria Burkholderia mallei Glanders (horses); potential bioterrorism a ent Burkholderia pseudomallei Melioidosis, Whitmore's disease, endemic in tro ical climates Burkholderia fungorum "Group", humand and plant pathogens and environmentall im ortant bacteria Neisseria menin hides Bacterial menin itis Neisseria onorrhoeae Gonorrhea Ralstonia solanaccarum Plant disease, "Southern wilt"

Ralstonia metallidurans Heav metal resistant Chlam dia muridarum Chlam dia - STD

Chlam dia trachomatis Chlam dia - STD

Chlam do hila neumoniae 10% of neumonia Chlam do hila sittaci Psittacosis arrot fever Chlorobium vibrio orme Green sulfur bacterium Clorobium to idum Green sulfur bacterium Rhodothermus marinus Hermo hillic halo hillic bacterium C to ha a hutchinsonii Di ests c stalline cellulose Shewanella oneidensis Can turn soluble metals insoluble.
Bioremediation Vibrio vulni rcus Warm seawater infects o en wounds Vibrio parahaemolyticus Warm seawater infects open wounds;
diarrhea Xanthomonas cam estris Plant atho en Xanthomonas axono odis Plant atho en Pirellula Plant atho en Brown algae Fucus vesiculosus Bladder wrack Advantageously, the composition of the present invention is effective to cure or prevent a disease caused by bacteria, archaea, and/or eucarya. The composition is effective to prevent formation of the 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 l0 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 2 0 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 2 5 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 morphein forms and in the amino acid sequence of the morphein surfaces. In the case of the more general inhibition of protein function through the selective stabilization of one morphein form, it may be the case that the target is a pathway that is not present in humans or it may be 3 0 the case that the target simply has sufficient phylogentic variation outside the active site that the surfaces of the morpheins 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 3 5 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 absorption 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 incorporated into the compositions.
Solutions of the 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.
2 0 The composition of the present invention is advantageously administered in the form of injectable 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 purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other 2 5 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 3 0 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, 3 5 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 of the 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 of the 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 of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the 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 2 0 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 of the present invention include both dilute compositions, which are ready 2 5 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 3 0 combination thereof. They may also be in the form of dispersible powders or grains, comprising a wetting agent to facilitate the dispersion of the 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 of the active ingredients in water optionally containing a surface-active agent, or may comprise a solution or 3 5 dispersion of the 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 agents) 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 multiineric porphobilinogen synthase that substantially exist in a multimeric form of a hugging dimer. In 2 0 certain embodiments, the multimeric porphobilinogen synthase is derived from a human. In certain embodiments, the multimeric porphobilinogen 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 porphobilinogen synthase.
The expression in a plant of a gene that exists in double-stranded DNA form involves 2 5 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 of the 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
3 0 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 of the protein encoded therein by the cells protein biosynthetic machinery.
In the instant invention, the promoter chosen will have the desired tissue and 3 5 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 l0 in the art.
In a preferred embodiment of the 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 (CTP)/PBGS) 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 alb binding protein gene promoters and the phytochrome promoter which has been utilized 2 0 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 2 5 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 3 0 sustain growth. In one embodiment, said promoter is leaky in order to provide tetrapyrroles necessary for the non-photosynthetic functions of the plant.
Plastid-directed Expression of PBGS Activity In a preferred embodiment of the 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 3 5 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-1,5-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 of the 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 S' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic 2 0 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 2 5 enhance expression of the 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 of the chimeric plant gene contains a polyadenylation signal 3 0 that functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the 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 of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene.
3 5 Plant Transformation/Regeneration In developing the nucleic acid constructs of this invention, the various components of the 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 of the 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 of the host cell.
A recombinant DNA molecule of the 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 (nptII) gene (Potrykus et al.,1985), which confers kanamycin resistance. Cells expressing the nptII gene can be selected using an appropriate antibiotic such as kanamycin or 6418. 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).
2 0 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, r 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, 3 0 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 of the following: a CTP; a light inducible promoter;
the PBGS gene; an intron that provides a splice site to facilitate expression of the gene, such as the Hsp70 intron (PCT Publication W093/19189); 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 fox 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 StuI-HindIII
fragment of pMON316 (Rogers et al., 1987) into pMON526. Plasmid pMON526 is a simple derivative of pMON505 in which the SmaI site is removed by digestion with XmaI, 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 2 0 for SmaI between the promoter and polyadenylation signal.
Binary vector pMON505 is a derivative of pMON200 (Rogers et al., 1987) in which the Ti plasmid homology region, LIH, has been replaced with_a 3.8 kb HindIII to SmaI 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 2 5 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 spectinomycinlstreptomycin resistance determinant for selection in E. coli and A. tumefaciens, an intact nopaline synthase gene for facile scoring of transformants 3 0 and inheritance in progeny, and a pBR322 origin of replication for ease in making large amounts of the vector in E. coli. Plasmid pMON505 contains a single T-DNA border derived from the right end of the 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 of the integrated DNA is 3 5 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 pMON17227. 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 ( I 990);
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 2 0 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:I):

cttacgcggtctgtgggagaccggagcgggagacagcggtgacaggagcagcggccgggagcccttagggaggcagaca gag cctgcagccaatgccccaggagccctcggttccaaccaactgatgcccctgtgcccactggcccacgccatgcagcccc agtccgtt ctgcacagcggctacttccacccactacttcgggcctggcagacagccaccaccaccctcaatgcctccaacctcatct accccatctt tgtcacggatgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtgaagcggctggaa gagatgct gaggcccttggtggaagagggcctacgctgtgtcttgatctttggcgtccccagcagagttcccaaggacgagcggggt tccgcag ctgactccgaggagtccccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcctgtga tgtctgcctg tgtccctacacctcccatggtcactgcgggctcctgagtgaaaacggagcattccgggctgaggagagccgccagcggc tggctga ggtggcattggcgtatgccaaggcaggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatc aaagag gccctgatggcacatggacttggcaacagggtatcggtgatgagctacagtgccaaatttgcttcctgtttctatggcc ctttccgggat gcagctaagtcaagcccagcttttggggaccgccgctgctaccagctgccccctggagcacgaggcctggctctccgag ctgtgga ccgggatgtacgggaaggagctgacatgctcatggtgaagccgggaatgccctacctggacatcgtgcgggaggtaaag gacaag caccctgacctccctctcgccgtgtaccacgtctctggagagtttgccatgctgtggcatggagcccaggccggggcat ttgatctcaa ggctgccgtactggaggccatgactgccttccgcagagcaggtgctgacatcatcatcacctactacacaccgcagctg ctgcagtg gctgaaggaggaatgatggagacagtgccaggcccaagaactagaactttaaaacgttcccggggcctcagacaagtga aaacca aagtaaatgctgcttttagaactgtgccctcatgccctcttcctgctcacatgctagcggggcccagcagccctgggtg gttttgccagc atgctaactcttgtaactcgcagctgcatcctatgagctctcccaagcttccccgcccctcccctgggtcagccgtgag gcccacctttg ccaccctcagctctttcctctggtgtggcttcagcttgaaagcaacctggagtcgggggcacagcctttggggcctggc tgggagag ggtcttggagcattaggggaagaagagagcagtgggatcttggggcctgagaagccttggaacgcttctggcagcagag ctgggt gtgggaatgaggcctagatcgatatccctgggttagagttgaaatttgccgcaattccactggaaggcatttcccacga ggccagagg ttgccaggctgcctgaggtctcctattctactctgaaccataaacccagagaagaattactcattaaccagcataaata ctgcctgagga tcaaaactcagaggcaaagagggagttcctgactgctagaggtgccaccaccacaaacactttttattcaggagatact ttttgagaatc tctgctctgttcctaggttcagtgctgggtcctgggaatacagcaggacagacctcagcttatctcttcatagaaatta tacaaagagaat tggggagacagctaagaagaaaacaaagaaataaagcagttacaaattgtgataagtgctttgaaggaaagaaggggtc tgagaca acaacagggaaggggcctctcttgaaacagtagttgggaaggaggcagacatgcaccagtgatgtggtgacaggtgctc tgaagg aggtcaccaggacctgacctctttgaaggatcagaaaatacttccctgaaggactgacatttgagcctagacctgaagg gtgagccat caagctaagacaattggggaagagcattccagggagagggaggagttgtgcaaaggccctggggctccttctagctgga ggaatg caaggctagcttgtctggagcactgagaggatggcctgaactgagtggagagagacagaccaggaccaaaccatgcaga ggtcaa gggccacattcaccttttcagagtgactcaatcaaatttgtagtttgtaaaagtattttaacagctctgcggcaaagtg caaatgaaaagtc ttgatggcatggactggagcggggacagtggggatggagaaaggggaatggattgtggatgtgtttagaaggtagattc gatgtgaa ggatgaatctggcttgaccttctgggtggctgatgggccatttactgagatggggcagcctggaagaggaacagaagca gggtcgg ggtggagggagaatactaaacttagcttgagacattttgcaataaggaagctatatctagagtgcttatgtgactcacc taaggccactc aacaagtttgtggcagaactggattagaactgcacagaaaacagccaagctgggatttgaacccatgtagtccaactcc aaggcctct gcccctaaccactgtgccataccacctcccaataatcaacagcaaaattataggtctaacaatgttttatagacacccc tccatttatgtga tgggtttgcatcctgataaacccatcataagttgaaaatatgatcataagttgaaaatatgatcataagtcaaaaatgt atttaatataccta acctaccaaacatcatagcttagcctagcctgccttaaacatgctcagaacacttacattagcctacagtgggcaaaac tatccaacaca aaatctatattgtaataaagttgtaaagaattttgaataaaaattcaatatttgaaaaaaaaaaaaaaaaa Human PBGS cDNA (SEQ ID N0:2):
gcagccaaagccccaggagccctaggttccaaccaactgatgcccctgtgcccactggcccacgccatgcagccccagt ccgttctgca cagcggctacttccacccactacttcgggcctggcagacagccaccaccaccctcaatgcctccaacctcatctacccc atctttgtcacgg atgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtgaagcggctggaagagatgct gaggcccttggt ggaagagggcctacgctgtgtcttgatctttggcgtccccagcagagttcccaaggacgagcggggttccgcagctgac tccgaggagtc cccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcctgtgatgtctgcctgtgtccc tacacctcccatggt cactgcgggctcctgagtgaaaacggagcattccgggctgaggagagccgccagcggctggctgaggtggcattggcgt atgccaagg caggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatcaaagaggccctgatggcacatgg acttggcaac agggtatcggtgatgagctacagtgccaaatttgcttcctgtttctatggccctttccgggatgcagctaagtcaagcc cagcttttggggacc gccgctgctaccagctgccccctggagcacgaggcctggctctccgagctgtggaccgggatgtacgggaaggagctga catgctcatg gtgaagccgggaatgccctacctggacatcgtgcgggaggtaaaggacaagcaccctgacctccctctcgccgtgtacc acgtctctgga gagtttgccatgctgtggcatggagcccaggccggggcatttgatctcaaggctgccgtactggaggccatgactgcct tccgcagagca -ggtgctgacatcatcatcacctactacacaccgcagctgctgcagtggctgaaggaggaatgatggaggacagtgcca ggcccaagaac tagaactttcaaacgttcccggggcctcagacaagtgacaaccaaagtaaatgctgcttttagaactgt Human PBGS amino acid sequence (SEQ ID N0:3):
MQPQSVLHSGYFHPLLRAWQTATTTLNASNLIYPIFVTDVPDDIQPITSLPGVARYGV
KRLEEMLRPLVEEGLRCVLIFGVPSRVPKDERGSAADSEESPAIEAIHLLRKTFPNLL

MDGRVEAIKEALMAHGLGNRVSVMSYSAKFASCFYGPFRDAAKSSPAFGDRRCYQ
LPPGARGLALRAVDRDVREGADMLMVKPGMPYLDIVREVKDKHPDLPLAVYHVS
GEFAMLWHGAQAGAFDLKAAVLEAMTAFRRAGADIIITYYTPQLLQWLKEE
4 5 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 andlor 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 of the skin, mucosa or hair of a person in need of said antimicrobial treatment with an antimicrobially effective amount of the compound of the 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.
2 0 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-2 5 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, mufti-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 3 0 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 3 5 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-2 0 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,semi-permanent_or permanent hair colourants, preparations containing self oxidising dyes, or natural hair colourants, such as henna or camomile.
2 5 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, 3 0 e.g. amine fluorides, which are known under the trade name Olafluor.
The compositions of the 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 3 5 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 purposes, may also be provided with anti-microbial 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-purpose 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.
2 0 The compositions of the 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 2 5 Further, the composition of the 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.
3 0 Further, the compositions of the 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 of the active form of the multimeric porphobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided 3 5 that the active form of the multimeric porphobilinogen 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 of the 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:
2 0 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 2 5 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 3 0 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 F 12L mutation) that dictated the dramatically different functional properties of F 12L
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 F 12L0 that were 35 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 F 12L variant was GGCTACCTCCACCCACTGCTTCGGGCC.
Several constructs were prepared for the coexpression of Wt and F 12L in E.
coli. Both the order of the 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 BamHI and NdeI to cut out Wt.
The pET17b vector DNA was linearized by digestion with BamHI and NdeI and ligated with Wt such that the ATG start codon of Wt was 6 basepairs downstream of the ribosomal binding site encoded by the vector. The resultant plasmid was transformed into E. coli XL 1 blue. Plasmid DNA (pETl7bWt) was prepared and linearized with SpeI and Bpu1102I. Plasmid DNA
containing the gene F12L (pET3aF12L) was digested with XbaI and Bpu1102I to produce a fragment containing the ribosomal binding site and the gene for F 12L. The gene for F 12L and the linearized pETl7Wt vector were ligated such that the ribosomal binding site of the F12L
gene was 3 5 basepairs downstream of the stopcodon of Wt, and the terminator was 52 basepairs downstream of the stop codon for the F12L gene. Plasmid pETl7bWtF12L was transformed into E.coli XLlblue, 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 2 5 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 of the 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 pM Zn(II), and employed a KCl gradient as shown in Fig. 3A. The gradient was controlled 3 0 by a Rainin HPLC system run at a flow rate of 3 ml min-1 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-mercaptoethanol, 10 ~M Zn. For the pH rate profiles, the reported pH
reflects the assay pH
after the addition of 10 mM ALA-HCI. For Km and Vmax determinations, concentrations of ALA were 10 pM, 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 l0 determine porphobilinogen formed.
ANALYTICAL ULTRACENTRIFUGATION:
Protein samples were dialyzed into 30 mM potassium phosphate, pH 7.5, 0.1 mM
DTT, and 10 pM ZnCl2 just prior to loading into the ultracentrifuge. Loading concentrations were 10.6 ~M and 12.8 ~M for wild type and F 12L 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 centerpieces using quartz windows. Data were collected at three rotor speeds (8,000, 11,000, and 14,000 rpm) 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 2 0 calculated using the Sednterp 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 of the data ruled out a single species as the residuals from the fits were clearly nonrandom.
2 5 A CRYSTAL STRUCTURE WAS DETERMINED FOR F 12:
F 12L was dialyzed against 50 mM bis-tris propane, 10 mM 13ME, and 10 p,pM
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 3 0 were collected at 100K on MAR345 image plate detector coupled with RU-200 rotating anode generator equipped with OSMIC optics and operated at SOkV and 100ma. Crystals were cryoprotected before freezing by transferring them at reservoir solutions containing 12%,17%, 23% 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 F 12L 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 a=b=89.6 ~, c=153.2 ~. 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 ~
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 of the 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 0.18 and 2.0°, respectively. All residues belong to allowed conformation regions on the Ramachandran plot.

Human PBGS variant F 12L is remarkably different from the wild type protein.
Characterization of purified F12L confirmed that the catalytic activity is very low under 2 0 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 F 12L 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 F 12L;
the results are presented in Table 1 (below). F 12L exhibits normal Michaelis-Menten kinetics with 2 5 extraordinarily high Km values, well above physiological concentrations of the substrate 5-aminolevulinic acid (ALA). However, at pH 9 the Vmax of F 12L is significantly higher than that of the 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 pM (6, 8-10), as is seen here for wild type human PBGS at pH 7.
3 0 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 (morpheins, 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 F 12L variant and the wild type protein came from variations in mobility during anion exchange chromatography (Fig. 1B) 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 of the 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 2 0 structure of human PBGS variant F 12L.

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 (L1-20) 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 a/(3 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 3 0 subunit interactions. The sequence of the 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 of the 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 of the tetramer, 3 5 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 a/(3 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 2A, 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 of the crystal structure of F 12L, 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 of the F 12L human PBGS
allele (PDB
Code 1PV8) reveals a quaternary structure that involves significant rearrangement of the N-terminal arm relative to the a/(3 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 a/(3 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 2 0 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 of the hugging dimer and the detached dimer. This interconversion process is 2 5 illustrated in Fig. SA.
The new structure of F 12L (2.2 A resolution) contains significant regions of disorder that impede a structural comparison of the 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 3 0 observed in both structures, most are superimposable. Thus, to further probe the basis for the unusual kinetic properties of F 12L (e.g., Fig. 1, Table 1 ), the inventor undertook coexpression of F 12L and wild type human PBGS.

QUATERNARY STRUCTURE IS THE BASIS FOR THE KINETIC DIFFERENCES:
A coexpression system was prepared to produce both wild type human PBGS and the F 12L 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. 3A). 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 of the heteromeric species. Pools I and II were further purified by gel filtration on Sephacryl 5300, which reduced cross contamination of the heteromers. The pH rate profiles of the 5300 purified Pools I and II are remarkably like F 12L
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 2 0 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 of the 5300 purified pools were determined at pH
7 and at pH 9 (Table 2). The kinetic data do not follow a simple Michaelis-Menten relationship 2 5 (hyperbolic fit), but can be attributed to catalysis by two different forms of the 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 of the 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 3 0 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.
3 5 Table 2 The kinetic parameters for wild type human PBGS, F 12L, and the pools of heteromeric Wt/F 12L
F12L; mld type, Wtfl2L ~ WtFl2L
n~:~ ~ s,. ... -_ ,_.., , ' Pool I~__.:;
Kml ~ ~: -~ 0.25+0.01 . : Pool pH 7 V 0.210.01 II-- -0.130.01 Vmaxl pH 7 55.5+0.2 2.370.07 20.200.99 Km2 pH 7 17.7+1.1 7.710.72 4.852.04 Vm~2 pH 7 1.14+0.05 4.780.10 10.9210.85 Kml pH 9 0.35+0.09 0.100.13 0.02410.003 Vmaxl pH 9 8.16+0.13 0.3210.23 3.190.15 Km2 pH.9 4.6 +0.1 4.46+0.80 3.740.29 2.350.21 Vm~2 pH 9 18.2+0.2 6.670.36 12.160.18 8.700.17 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. Km 1 and Km2 (both mM) are interpreted as the Km for the octamer and hexamer, respectively. The reported Vm~ values (in units of pmoles h-1 mg-1 ) reflect the mole fraction of quaternary species under assay conditions, which remains to be determined.
Fitted Km values are independent of the distribution of quaternary species.
The data presented on wild type human PBGS, the F 12L variant, and the WT+F

heteromers definitively establishes that the kinetic differences between the wild type protein and the F 12L 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 F 12L and that the kinetics of the octamer are like that of the wild type protein.
In light of the 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 of the 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 2 0 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 F 12L is lacking in density from most of the 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 of the 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 of the 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 l0 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 of the 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 arrri-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. 4A shows the 2 0 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 of the 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 of the allosteric 2 5 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 3 0 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 of the allosteric magnesium causes the Km value to decrease from ~2 mM to 200 Mm (8), which is remarkably reminiscent of the difference between the Km values of the 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.
1 o 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 y-proteobacter), B.
japonicum (an a-proteobacter), P. aeruginosa (a y-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) 2 0 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 F 12L leads us to propose that the 2 5 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. SA. 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 3 0 favor formation of the hugging-dimer and, hence, the octamer. Fig. SB
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 of the 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 regularis (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 2 0 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 2 5 allosteric regulation of protein function.
This invention describes inhibition of protein function through stabilization of the inactive morphein of PBGS and/or any other protein that might be regulated by the interconversion of morpheins. 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 3 0 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 PEGS, the difference between octamer and hexamer lies in the structure of the 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 oftarget 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) JMoI 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.e~asy.ch/spdbvlmain~ag_e.html) and other programs. To build the P.
aeruginosa PBGS
2 0 hexamer, the N-terminal arms were removed from the structure file for the 1 GZG dimer. The resulting alpha, beta-barrel domains (residues 32-335) were successively overlaid upon the three dimers of hexameric1 PV8 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 of the N-terminal arm.
2 5 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 Natl Acad Sci U S A 99, 7432-7437) was used to model amino acids 29-32, so as to connect the N-terminal 3 0 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 of the N-terminus of the human PBGS
hexamer (1 PV8), the hexamer model for P. aeruginosa PBGS is missing residues 1-9 of subunits A, C, and E as 3 5 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) JBiol 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 of the 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 of the 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 of the alpha-beta-barrel of the 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 morphein 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 2 0 bind to the "arm pit" of the 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, ~-[[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propenyl]oxy]-3,4-dihydroxy-, (DR)- (9CI)) is consistent with a 2 5 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 pg/ml PBGS. This means that at 3.5 pg/ml, under assay conditions, the equilibrium of quaternary isoforms (morpheins) contains about 50% octamer and 3 0 about SO% 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 morphein 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 ~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.
16A show the protein concentration dependence of the 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. Thus, following this treatment with rosmarinic acid, 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. This is consistent with the interpretation 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 2 0 octamer and hexamer (see also FIG SB). Lanes 1 and 3 show that the equilibrium is shifted to smaller forms 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 of the addition of 10 mM substrate on this quaternary structure equilibrium.
Based on our modeling results, the interactions of this biphenyl compound with the "arm 2 5 pit" of the 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 of the rosmarinic acid. Hence, a derivative of the 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 3 0 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 of the propanoic acid portion of the 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 3 5 made therein without departing from the spirit and scope thereof.

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Biol Chem 278, 31325-31330) 3 0 93. PCT Publication WO 84/02913 94. PCT Publication W093/19189 95. PCT Publication WO 92/04449 SEQUENCE LISTING
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<213>
Homo Sapiens <400>

gcagccaaagccccaggagccctaggttccaaccaactgatgcccctgtgcccactggcc60 cacgccatgcagccccagtccgttctgcacagcggctacttccacccactacttcgggcc120 tggcagacagccaccaccaccctcaatgcctccaacctcatctaccccatctttgtcacg180 gatgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtg240 aagcggctggaagagatgctgaggcccttggtggaagagggcctacgctgtgtcttgatc300 tttggcgtccccagcagagttcccaaggacgagcggggttccgcagctgactccgaggag360 tccccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcc420 tgtgatgtctgcctgtgtccctacacctcccatggtcactgcgggctcctgagtgaaaac480 ggagcattccgggctgaggagagccgccagcggctggctgaggtggcattggcgtatgcc540 aaggcaggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatc600 aaagaggccctgatggcacatggacttggcaacagggtatcggtgatgagctacagtgcc660 aaatttgcttcctgtttctatggccctttccgggatgcagctaagtcaagcccagctttt720 ggggaccgccgctgctaccagctgccccctggagcacgaggcctggctctccgagctgtg780 gaccgggatgtacgggaaggagctgacatgctcatggtgaagccgggaatgccctacctg840 gacatcgtgcgggaggtaaaggacaagcaccctgacctccctctcgccgtgtaccacgtc900 tctggagagtttgccatgctgtggcatggagcccaggccggggcatttgatctcaaggct960 gccgtactggaggccatgactgccttccgcagagcaggtgctgacatcatcatcacctac1020 tacacaccgcagctgctgcagtggctgaaggaggaatgatggaggacagtgccaggccca1080 agaactagaactttcaaacgttcccggggcctcagacaagtgacaaccaaagtaaatgct1140 gcttttagaactgt 1154 :210> 3 :211> 330 :212> PRT
:213> Homo Sapiens ;400> 3 Met Gln Pro Gln Ser Val Leu His Ser Gly Tyr Phe His Pro Leu Leu 1 5 10 r 15 Arg Ala Trp Gln Thr Ala Thr Thr Thr Leu Asn Ala Ser Asn Leu Ile Tyr Pro Ile Phe Val Thr Asp Val Pro Asp Asp Ile Gln Pro Ile Thr Ser Leu Pro Gly Val Ala Arg Tyr Gly Val Lys Arg Leu Glu Glu Met Leu Arg Pro Leu Val Glu Glu Gly Leu Arg Cys Val Leu Ile Phe Gly 65 70 75 gp Val Pro Ser Arg Val Pro Lys Asp Glu Arg Gly Ser Ala Ala Asp Ser 85 90 g5 Glu Glu Ser Pro Ala Ile Glu Ala Ile His Leu Leu Arg Lys Thr Phe Pro Asn Leu Leu Val Ala Cys Asp Val Cys Leu Cys Pro Tyr Thr Ser His Gly His Cys Gly Leu Leu Ser Glu Asn Gly Ala Phe Arg Ala Glu Glu Ser Arg Gln Arg Leu Ala Glu Val Ala Leu Ala Tyr Ala Lys Ala Gly Cys Gln Val Val Ala Pro Ser Asp Met Met Asp Gly Arg Val Glu Ala Ile Lys Glu Ala Leu Met Ala His Gly Leu Gly Asn Arg Val Ser Val Met Ser Tyr Ser Ala Lys Phe Ala Ser Cys Phe Tyr Gly Pro Phe Arg Asp Ala Ala Lys Ser Ser Pro Ala Phe Gly Asp Arg Arg Cys Tyr Gln Leu Pro Pro Gly Ala Arg Gly Leu Ala Leu Arg Ala Val Asp Arg Asp Val Arg Glu Gly Ala Asp Met Leu Met Val Lys Pro Gly Met Pro Tyr Leu Asp Ile Val Arg Glu Val Lys Asp Lys His Pro Asp Leu Pro Leu Ala Val Tyr His Val Ser Gly Glu Phe Ala Met Leu Trp His Gly Ala Gln Ala Gly Ala Phe Asp Leu Lys Ala Ala Val Leu Glu Ala Met Thr Ala Phe Arg Arg Ala Gly Ala Asp Ile Ile Ile Thr Tyr Tyr Thr Pro Gln Leu Leu Gln Trp Leu Lys Glu Glu

Claims (52)

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 of the 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 porphobilinogen synthase and a Class 1a ribonucleotide reductase.

8. The composition of claim 7, wherein said multimeric protein is porphobilinogen synthase comprising eight porphobilinogen 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 porphobilinogen synthase monomers.

10. The composition of claim 7, wherein said multimeric protein is the Class Ia ribonucleotide reductase and the agent inhibits the Class Ia 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 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.

12. The composition of claim 11, wherein the multimeric porphobilinogen synthase is derived from bacteria, archaea, or eucarya, provided that the octameric porphobilinogen synthase contains an allosteric magnesium binding site.

13. The composition of claim 12, wherein the multimeric porphobilinogen synthase contains a catalytic zinc binding site.

14. The composition of claim 11, wherein the multimeric porphobilinogen 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 of the active form of the multimeric porphobilinogen synthase, said active form is an octomeric porphobilinogen synthase by binding to a hug-disabling domain of the less active form of the multimeric porphobilinogen 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 porphobilinogen 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 of the active form of the multimeric porphobilinogen 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 of the active form of the multimeric porphobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric porphobilinogen 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 of the active form of the multimeric porphobilinogen synthase and thereby inhibiting or preventing development or growth of bacteria, archaea, and/or eucarya, provided that the active form of the multimeric porphobilinogen 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 porphobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.

32. The herbicide resistant plant of claim 31, wherein the multimeric porphobilinogen synthase is derived from a human.

33. The herbicide resistant plant of claim 31, wherein the multimeric porphobilinogen synthase contains no allosteric magnesium binding site.

34. A composition comprising an inhibitor adapted to bind to a multimeric porphobilinogen 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 porphobilinogen synthase and a Class Ia ribonucleotide reductase.

44. The method of claim 43, wherein said multimeric protein is porphobilinogen synthase comprising eight porphobilinogen synthase monomers.

45. The method of claim 43, wherein said multimeric protein is the Class Ia ribonucleotide reductase and the agent inhibits the Class Ia 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 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 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 porphobilinogen synthase from forming an active form, the method comprising:
applying the composition of claim 11 to the multimeric porphobilinogen 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 porphobilinogen 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 porphobilinogen synthase that substantially exist in a multimeric form of a hugging dimer.

49. The method of claim 48, wherein the multimeric porphobilinogen 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.