CN1835739A - 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

Six aggressiveness porphobilinogen synzyme are as the target of exploitation antibiotic and herbicide

Cross reference to related application

The application requires the priority of provisional application of submitting on July 7th, 2,003 60/485,253 and the provisional application of submitting on June 4th, 2,004 60/577,312, more than two applications to draw in full with it at this be reference.

To quoting of material in the CD

Being stored in that sequence table in the CD draws with it in full at this is reference.

Background of invention

1. invention field

The present invention relates to the biosynthesis of tetrapyrrole, and more specifically relate to the mechanism that suppresses the activation of porphobilinogen synzyme.

2. description of Related Art

The tetrapyrrole biosynthesis is an animal, and plant and microorganism comprise antibacterial, archeobacteria, fungus, the critical path of protista and virus.Modal intermediate product is 5-aminolevulinic acid (ALA).Enzyme reaction in all organisms from ALA to the protoporphyrin IX is common for the biosynthesis of tetrapyrrole.

Porphobilinogen synzyme (PBGS) is also referred to as 5-aminolevulinic acid dehydratase (ALAD), is ancient and albumen high conservative, and its catalysis tetrapyrrole comprises haemachrome, chlorophyll, vitamin B ₁₂With cofactor F ₄₃₀The modal step of (1,2) biosynthesis.The condensation of two 5-aminolevulinic acids of PBGS catalysis molecule forms tetrapyrrole precursor porphobilinogen.

PBGS was understood that it is homotype eight aggressiveness metalloenzyme in the past, and it uses multiple bivalence or monovalent cation in catalytic site and allosteric site.As if mammal and yeast need Zn (II) usually, and some procaryotic enzyme require Mg (II) or Zn (II) or both are reaching maximum activity, and the enzyme of plant only needs Mg (II) to realize enzymatic activity.A spot of organism has neither needs Zn (II) also not need the PBGS of Mg (II).It is caused by the variation of residue in the primary structure at least two melts combine zones using the difference of metal ion.

The specific activity that has been found that the PBGS in some source depends on protein concentration.For example the living slowly root nodule bacteria of Japan (B.japonicum), Pseudomonas aeruginosa (P.aeruginosa), with the active protein concentration dependency of visual contrast among the Semen Pisi sativi PBGS, but also not about from escherichia coli (E.coli), yeast, or the document of the PBGS in mammal source (22) proves.

Known to removing metal from avtive spot, for example PBGS is handled with ethylenediaminetetraacetic acid (EDTA) and can suppress PBGS.

More information about PBGS is disclosed in list of references (3-5) and (7).

At present, more consumers requires the personal health care product such as wet tissue, the function that diaper etc. can not only provide them to want, can also treat or prevent owing to contact for example antibacterial territory, the archeobacteria territory, and/or eukaryote territory (eucarya) and the i or I that causes, do not damage the health of consumer simultaneously.For satisfying such demand, for example mix antimicrobial in the wet tissue, to persist or resident antibacterial on the opposing skin in the consumer products of broad range.The product that contains antimicrobial occurs in market with a lot of forms at present, lotion for example, the flavor soap of dispelling, hard surface cleaner, wet tissue and surgery disinfectant.

Yet many products that contain antimicrobial owing to be used to provide anti-microbial effect chemicals character and skin is produced injury or stimulates.For example, some hard surface cleaners and surgery disinfectant are used high levels of alcohol and/or surfactant, and it has been repeated to show dry also generation of skin histology stimulated.Other present available wet tissue use harsh cationic surfactant and do not add acid.Although the surfactant energy is deep enough and kill polytype antibacterial, it produces skin stimulates and injury.

Many products that contain antimicrobial use organic acid, make up as antimicrobial with anion or cationic surfactant.Although some organic acid can need not surfactant and be used for product safely with controlling microbial, it is not good that great majority only mix organic acid product anti-bacterial effect, unless use very high concentration.Under high concentration very, these acid make that last product is uneconomical and even cause skin irritant problem.

In addition, biomembrane also is the problem that some surface exists.Biomembrane can be found on the environmental surfaces of the abundant dampness of any existence basically.Their speed with fastest developing speed in the running system that enough nutrients can get.Biomembrane is made of microbial population that is adsorbed onto environmental surfaces or group, and it is the aggregation of cell and polysaccharide complexity.These microorganisms are enclosed in their synthetic extracellular polysaccharides usually.This biomembrane for example can be from Pseudomonas aeruginosa, and the mixed culture of pseudomonas fluorescens (P.fluorescens) and klepsiella pneumoniae (Klebsiella pneumonia) forms.Biomembrane can be gone up in the solid basic unit (substrate) that contacts with dampness and form, and forms in the soft tissue surfaces formation of live organism with at the liquia air interface.Produce rock and other stromal surface that the common position of biomembrane comprises ocean or fresh water environment.Biomembrane is also relevant with live organism usually, comprises plant and animal.Tissue surface for example is bathed in the complicated aggregation of the tooth that enriches water-bearing media and the microorganism of the fast-developing peplos of mucous membrane of small intestine in the extracellular polysaccharide of their own generations lastingly.Oral cavity bacterium is relevant with the tooth cavity in the ability that its cell interior stores close iodine polysaccharide or glycogen sample molecule, because these storage compounds can make the time lengthening that may form lactic acid.The time lengthening that is exposed to lactic acid has just caused enamel decalcification.

People utilize microbial biofilm, mainly remedy in the district in the habitat.Water treatment plant, wastewater treatment plant and the corrupt system relevant with individual family by with biomembrane interact remove pathogen and reduce water or waste water in the content of organic substance.On the other hand, biomembrane can be the serious harm to health, especially in the patient who introduces artificial basic unit.In addition, biomembrane is the threat to the wheel boat bottom, because barnacle can and corrode this surface or the pipeline inwall of oil pump or dehumidifier for example in its superficial growth.

Although aforesaid development is arranged, for providing antimicrobial compositions that demand is still arranged with widespread usage.Also need to provide and to disturb the equilibrated reagent in polymer protein unit, for example, can suppress the biosynthetic inhibitor of tetrapyrrole in plant and/or the antibacterial.Also need to finish inhibition, thereby produce a kind of method of inhibition antibacterial, antibiosis or herbicidal activity new, high special by the mechanism that is not suitable for the human or animal.

All lists of references cited herein are with its hereby incorporated by reference.

The invention summary

Therefore, the invention provides a kind of combination of agents thing that comprises, thereby this reagent by being attached to multimeric protein binding site and influence unitary balance and influence multimeric protein, wherein said multimeric protein comprises having a plurality of described unitary set, wherein each described unit comprises that first complementary surface is relevant with another unitary second complementary surface with second complementary surface and unitary first complementary surface, suppose that this set is at least a following different quarternary structure isoform, condition is the structure of the described different quarternary structure isoform of (1) each described unitary structures shape in the described multimeric protein, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quarternary structure isoforms.In certain embodiments, influence the formation that described multimeric protein comprises influences the quarternary structure isoform.

In certain embodiments, influence the function that described multimeric protein comprises influences multimeric protein.The nonrestrictive example of described multimeric protein function is active, and wherein influence be meant suppress or activate at least a.

In certain embodiments, this reagent is attached to and has more SA quarternary structure isoform or have at least a in the more highly active quarternary structure isoform.

In certain embodiments, each described unit is selected from monomer, dimer, trimer, the tetramer, six aggressiveness and eight aggressiveness.

In certain embodiments, described multimeric protein is selected from porphobilinogen synzyme and Ia type ribonucleotide reductase.

In certain embodiments, described multimeric protein is to comprise eight monomeric porphobilinogen synzyme of porphobilinogen synzyme.In other embodiments, the activity form of polymer porphobilinogen synzyme has and is less than eight monomers.

In certain embodiments, this reagent is to be attached to the inhibitor with more SA quarternary structure isoform, and wherein the quarternary structure isoform contains and is less than eight porphobilinogen synzyme monomers.In certain embodiments, inhibitor is rosmarinic acid (rosmarinic acid) or derivatives thereof.

In certain embodiments, described multimeric protein is an Ia type ribonucleotide reductase, and this reagent suppresses Ia type ribonucleotide reductase by being selectively bound to the binding site with more SA quarternary structure isoform uniqueness.

A kind of compositions also is provided, it contains inhibitor, this inhibitor is by being attached to the formation that suppresses to have the monomeric polymer porphobilinogen of first quantity synthase activity form than the low activity form with the monomeric polymer porphobilinogen of second quantity synzyme, and wherein the first quantity monomer is more than the second quantity monomer.

In certain embodiments, polymer porphobilinogen synzyme is derived from the antibacterial territory, and archeobacteria territory or eukaryote territory suppose that eight aggressiveness porphobilinogen synzyme comprise the magnesium binding site of allosteric.In a variant of this embodiment, polymer porphobilinogen synzyme comprises catalytic zinc binding site.

In certain embodiments, polymer porphobilinogen synzyme does not comprise the magnesium binding site and the catalytic zinc binding site of allosteric.

In certain embodiments, described is six aggressiveness than the low activity form.In certain embodiments, wherein said is dimer than the low activity form.In certain embodiments, the activity form of polymer porphobilinogen synzyme is eight aggressiveness.

In certain embodiments, thus inhibitor substituted metal ion is combined in the metal ion binding site place.In certain embodiments, metal ion is zinc and/or magnesium.

In certain embodiments, inhibitor is combined in avtive spot.

In certain embodiments, inhibitor is not a metal cation.

In certain embodiments, inhibitor suppresses the formation of polymer porphobilinogen synthase activity form, and described activity form is to be less than eight the monomeric eight aggressiveness porphobilinogen synzyme of domain that can not implement to embrace (hug-disabling) than low activity form polymer porphobilinogen synzyme by being attached to contain.

In certain embodiments, inhibitor suppresses the formation of polymer porphobilinogen synthase activity form by being combined in site beyond avtive spot and/or the metal ion binding site.

In certain embodiments, inhibitor is by removing the formation of the mechanism inhibition polymer porphobilinogen synthase activity form outside the metal ion.

In certain embodiments, said composition also comprises delivery media, and described delivery media is selected from the medicine acceptable medium, oral acceptable carrier, antibacterium medium and effective weeding medium.

Advantageously, said composition effectively suppresses or prevents the formation of the activity form of polymer porphobilinogen synzyme, thereby and suppress or prevent antibacterial, archeobacteria and/or Eukaryotic growth or growth suppose that the activity form of polymer porphobilinogen synzyme contains the magnesium binding site of allosteric.In a variant of the present embodiment, compositions is effectively treated or is prevented because the disease that contact antibacterial, archeobacteria and/or eukaryote cause.In a variant of the present embodiment, compositions is at least a in medicine, toothpaste, soap, disinfectant, antibiont film composition and the herbicide.

In certain embodiments, thereby said composition effectively suppresses or the formation of the activity form of prevention polymer porphobilinogen synzyme and inhibition or pre-bacteriological protection, archeobacteria and/or Eukaryotic growth or growth suppose that the activity form of polymer porphobilinogen synzyme does not contain the magnesium binding site and the catalytic zinc of allosteric.In a variant of the present embodiment, said composition is effectively treated or is prevented by contact antibacterial, the disease that archeobacteria and/or eukaryote cause.In a variant of the present embodiment, said composition is at least a in medicine, toothpaste, soap, the disinfectant.

The plant of antiweed also is provided, and it is to being genetically modified to embrace the polymer porphobilinogen synzyme that dimeric polymer form exists mainly.In certain embodiments, this polymer porphobilinogen synzyme is from the people.In certain embodiments, this polymer porphobilinogen synzyme does not contain the magnesium binding site of allosteric.

A kind of compositions also is provided, and it comprises the inhibitor that is attached to the polymer porphobilinogen synzyme that does not need zinc realization catalysis.

The method that influences multimeric protein also is provided, this method comprises: provide to comprise the multimeric protein with a plurality of unit sets, wherein each described unit comprises that first complementary surface is relevant with another unitary second complementary surface with second complementary surface and one of them unitary first complementary surface, suppose that this set is at least a in the different quarternary structure isoforms, condition is the structure of the described different quarternary structure isoform of (1) each described unitary structures shape, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quarternary structure isoforms; The compositions of the present invention that comprises reagent is provided, and wherein this reagent influences balance by the binding site that is attached in this set; And should gather with reagent and contact, wherein this reagent influences balance by being attached to binding site and influencing described multimeric protein thus.In some embodiment of this method, influence the formation that described multimeric protein comprises influences the quarternary structure isoform.In some embodiment of this method, influence the function that described multimeric protein comprises influences described multimeric protein.

In some embodiment of this method, the unit is to be selected from monomer, dimer, trimer, the tetramer, six aggressiveness and eight aggressiveness.

In some embodiment of this method, the function of the described multimeric protein of agents influence.

In some embodiment of this method, the function of described multimeric protein be active and wherein influence be suppress or activation at least a.

In some embodiment of this method, reagent is incorporated at least a in the quarternary structure isoform that has more SA quarternary structure isoform or have greater activity.

In some embodiment of this method, reagent is attached to the quarternary structure isoform with greater activity.

In some embodiment of this method, described multimeric protein is selected from porphobilinogen synzyme and Ia type ribonucleotide reductase.

In some embodiment of this method, described multimeric protein is to comprise eight monomeric porphobilinogen synzyme of porphobilinogen synzyme.

In some embodiment of this method, described multimeric protein is an Ia type ribonucleotide reductase, and reagent suppresses Ia type ribonucleotide reductase by being selectively bound to the binding site with more SA quarternary structure isoform uniqueness.

The adjusting cell also is provided, the method of tissue or organism physiologically active, this method comprises: cell is provided, tissue and organism, cell wherein, tissue or the multimeric protein that comprises of organism contain and have a plurality of unitary set, wherein each unit comprises that first complementary surface is relevant with another unitary second complementary surface with second complementary surface and unitary first complementary surface, suppose that this set is at least a in the different quarternary structure isoforms, condition is the structure of the described different quarternary structure isoform of (1) each described unitary structures shape, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quarternary structure isoforms; And provide compositions of the present invention to cell, tissue or organism, and said composition comprises reagent, wherein this reagent influences balance by being attached to the binding site on the unit and influencing the formation of quarternary structure isoform thus and adjust physiologically active.

Also provide and suppress the method that polymer porphobilinogen synzyme forms activity form, this method comprises: compositions of the present invention is applied to polymer porphobilinogen synzyme; With compositions with link to each other than the low activity form; Thereby inhibition forms activity form than the assembling of low activity form and suppresses polymer porphobilinogen synzyme and forms activity form.

The method of handling plant growing or growth also is provided, comprise and to be administered to plant as the present composition of herbicide, wherein this plant is a Herbicid resistant, and to being genetically modified to embrace the polymer porphobilinogen synzyme that dimeric polymer form exists substantially.In a variant of this method, polymer porphobilinogen synzyme does not contain the magnesium binding site of allosteric.

The method for preparing the antibacterium surface also is provided, this method comprises: (1) provides compositions of the present invention, wherein thereby said composition effectively suppresses or prevents the formation of polymer porphobilinogen synthase activity form and suppress or prevent antibacterial, archeobacteria and/or Eukaryotic growth or growth, and it is at least a in medicine, toothpaste, soap, disinfectant, antibiont film composition and the herbicide that the activity form of supposing polymer porphobilinogen synzyme comprises the magnesium binding site of allosteric and said composition; (2) provide formation surface matrix (matrix); (3) compositions and surface are formed substrate and combine, thus and preparation antibacterium surface.In a variant of this method, the antibacterium surface prevents and suppresses biomembranous formation.

The accompanying drawing summary

The present invention describes in conjunction with following accompanying drawing, wherein gives same label to same element, wherein:

Figure 1A shows the pH variation diagram of wild type people PBGS with respect to the F12L variant.

Figure 1B is presented at chromatographic isolation wild type people (Wt) PBGS and F12L on the mono-Q post.

Fig. 1 C demonstration wild type (Wt) people PBGS and F12L are in the swimming difference of 12.5% polyacrylamide native gel electrophoresis.

Fig. 2 A shows the sketch map of dimer, the tetramer and eight aggressiveness of wild type people PBGS.

Fig. 2 B shows the sketch map of dimer, the tetramer and six aggressiveness of people PBGS variant F12L.

Fig. 3 A shows that proteic two peaks of PBGS are at Q-Sepharose (KCl gradient (---), A280 (-)) on chromatographic isolation.

Two ponds of Fig. 3 B demonstration Wt and F12L are the swimming difference in native gel electrophoresis with respect to wild type (Wt) people PBGS and F12L.

Fig. 3 C is presented at and is further purified back Wt and the pond I (●) of F12L and the pH variation diagram of pond II (■) on the Sephacryl S300.

Fig. 3 D shows the chart of the activity of ALA to concentration, is used for determining the pond I (circle) of S300 purification and pond II (square) K at pH 7 (black) and pH 9 (Lycoperdon polymorphum Vitt) _mAnd V _MaxValue.

Fig. 4 A shows the sketch map of escherichia coli (E.coli) PBGS crystal structure, comprises the position of the magnesium of allosteric.

Fig. 4 B shows the sketch map of people's PBGS eight aggressiveness (left side) with respect to the subunit interface of six aggressiveness (right side).

Fig. 5 A shows the equilibrated sketch map that exists between dimer, six aggressiveness and eight aggressiveness PBGS.

Fig. 5 B is presented at isolating Semen Pisi sativi PBGS under the situation that has magnesium, native gel electrophoresis under the situation of analysis condition and the increase of EDTA concentration.

Fig. 6 shows according to the magnesium that has allosteric (Mg) (grid zone) there is not the magnesium (white portion) of allosteric, has avtive spot zinc (Dark grey zone) and does not have the classification of the independent isolating standard of avtive spot zinc (white portion) to PBGS.The matrix that obtains (the rightest) is made up of four quadrants, Northwest Quadrant (NW quadrant) representative+Zn/-Mg wherein, Northeast Quadrant (NE quadrant) representative-Zn/-Mg, Southwest Quadrant (SW quadrant) representative+Zn/+Mg, and southeast quadrant (SE quadrant) representative-Zn/+Mg.

Fig. 7 A shows the comparison of eukaryote and archeobacteria PBGS sequence avtive spot melts combine residue, and the genome at GenBank and other network retrievals obtains but sequence is in April, 2002.

Fig. 7 B shows the comparison of eubacteria PBGS sequence avtive spot melts combine residue, and the genome at GenBank and other network retrievals obtains but sequence is in April, 2002.

Fig. 8 is the classification that comprises antibacterial, archeobacteria and Eukaryotic PBGS source, wherein basic of distribution Fig. 6 of the melts combine character of PBGS coding.

Fig. 9 A shows the dimeric axonometric chart of E.coli PBGS, and wherein protein protomer is shown as strip-chart.The avtive spot zinc ion is shown as light gray chromosphere (sphere), and the magnesium ion of allosteric is shown as black ball.

Fig. 9 A shows the dimeric axonometric chart of E.coli PBGS, and wherein protein protomer is shown as strip-chart and with black and light-grey colouration.The avtive spot zinc ion ball (sphere) that appears dimmed, and the magnesium ion of allosteric is shown as black ball.The avtive spot part does not show.

Fig. 9 B shows the axonometric chart of the CONSTRUCTED SPECIFICATION of avtive spot zinc.The cysteine part is labeled and the sulphur atom of cysteine is shown as white ball.Water is labeled.Avtive spot part 4,7-DOSA is shown as Lycoperdon polymorphum Vitt, and oxygen atom is spherical.

The axonometric chart of the CONSTRUCTED SPECIFICATION of the magnesium binding site of Fig. 9 C demonstration allosteric.Archon is illustrated in the hydrone that forms the connection network that extends between the magnesium of contiguous residue and oxygen and the nitrogen-atoms.The aminoacid that is included in this network is shown as clavate, and the color of carbon is shallow or dark according to the chain of Fig. 9 A.Being included in the oxygen or the nitrogen-atoms that connect in the network shows with correlated shade.The aminoacid E231 of labelling is unique aminoacid in first coordination sphere of magnesium.R11 is from the N-terminal arm of embracing the adjacent subunit of dimer.

Figure 10 shows the sketch map of the embodiment of process of inhibition of the present invention, and inhibitor wherein of the present invention (being represented by circle) is attached to one or more domains of dimer or six aggressiveness PBGS to suppress to form eight aggressiveness, the form of stable bond and shifting balance.

Figure 11 is an equilibrated two dimensional representation between proteic two isoforms, and the reagent that its demonstration can influence protein function a kind ofly has binding site in form and do not have at another kind in form unitary.In both cases, the rule of multimerization is with a thick line and a dotted line and puts.

Figure 12 is an equilibrated two dimensional representation between proteic two isoforms, and it shows that balance must be able to experience the exchange of different units.

Figure 13 is the equilibrated two dimensional representation between multiple quarternary structure isoform and unit and the oligomer.It shows four kinds of protein protomer not isomorphism types (multimeric protein).Multimeric protein can be dimer (being shown as ellipse herein), trimer (being shown as sphere), and the tetramer (being shown as square).The shape of unitary shape control multimeric protein for example, has to form dimer, trimer and tetrameric unit; In addition, also have can not oligomerization the unit.This figure display unit must experience the specific oligomer (isoform) of configuration conversion to form certain shape.In all cases, monomeric structure has been stipulated the state of oligomer, and the state of oligomer has been stipulated functional character.In all cases, must utilize unitary two kinds not isomorphism type with balance between polymer.

Figure 14 is an equilibrated two dimensional representation between unit and the oligomer that comprises allosteric regulatory factor (reagent).The allosteric regulatory factor is shown as the grey body of the filling that is attached to unit or multimeric protein.The allosteric regulatory factor can disturb the balance between the oligomer state.

Figure 15 shows as S756393 and is fitted to structure (space filling) in six aggressiveness Pseudomonas aeruginosa (P.aeruginosa) the PBGS models.

(unfilled circle forms the curve (Kd=3.5 μ g/ml) that does not use inhibitor and the curve (Kd=13.5 μ g/ml) of the circle formation use rosmarinic acid of filling to the A555 value that Figure 16 A measured in different analysis times.

Figure 16 B shows the figure (unfilled circle form the circle that do not use the curve of rosmarinic acid and fill form the curve that use 62.5mM rosmarinic acid) of specific activity to inhibitor concentration.

Figure 16 C demonstration inhibition is relevant with the less oligomer form of stable Semen Pisi sativi PBGS, and (10mg/ml Semen Pisi sativi PBGS is with rosmarinic acid preincubate 30min.IC50＝62.5mM)。

Detailed Description Of The Invention

The present invention is from the research of inventor to tetrapyrrole biosynthesis basic science. The invention provides and consider different oligomerization states, for example those oligomerization states relevant with signal and cell cycle regulating are the new methods that how can raise or reduce approach. According to the present invention, any allosteric is regulated the albumen that can define by the balance between the morpheins, and its activity can be incorporated into also reagent (such as the little molecule) adjusting of the balance of mobile quaternary structure form of unique surface feature of one or the other morpheins.

According to certain embodiments of the present invention, the tetrapyrrole biosynthesis can be regulated by the balance between the morpheins that regulates PBGS. According to certain embodiments of the present invention, inhibitor molecules can be found its preferably with the unique surface component interaction of PBGS six aggressiveness, and replace morpheins to the distribution (it is considered to inactive form in plant and some bacterium PBGS) of six aggressiveness forms.

Advantageously, the invention provides a kind of composition, thereby it balance that comprises the binding site by being attached to multimeric protein and affect the unit affects the reagent of multimeric protein, wherein said multimeric protein comprises the set with a plurality of described unit, wherein each described unit comprises that the first complementary surface of the first complementary surface and the second complementary surface and one of them unit is relevant with the second complementary surface of another unit, suppose that this set is at least a in the different quaternary structure isoforms, condition is the structure of the described different quaternary structure isoform of the structures shape of (1) each described unit in described multimeric protein, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quaternary structure isoforms. Composition of the present invention can be used for suppressing or preventing that bacterium, archeobacteria and/or eucaryote are in human or animal host's growth or growth. For example, composition of the present invention can be used for medicine, toothpaste, soap, disinfectant, antibiont film composition and herbicide form. The invention provides and select the target organism and affect this organism to obtain the guide of required effect.

The unit of multimeric protein can be, for example monomer, dimer, tripolymer, the tetramer, six aggressiveness and eight aggressiveness. In certain embodiments, affect the formation that described multimeric protein comprises affects the quaternary structure isoform. In certain embodiments, affect the function that described multimeric protein comprises affects described multimeric protein. The nonrestrictive example of described multimeric protein function be active and wherein impact be suppress or activation at least a. As mentioned below, according to the application, reagent can be incorporated at least a in the quaternary structure isoform that has more SA quaternary structure isoform or have greater activity, thereby suppresses or the activation multimeric protein.

Exemplary multimeric protein is porphobilinogen synzyme and Ia type ribonucleotide reductase. Therefore, in certain embodiments, this reagent is to be attached to the inhibitor with more SA quaternary structure isoform, and wherein the quaternary structure isoform contains and is less than eight porphobilinogen synzyme monomers. Similarly, when multimeric protein is Ia type ribonucleotide reductase, this reagent suppresses Ia type ribonucleotide reductase by being selectively bound to the binding site than low activity quaternary structure isoform uniqueness.

The present invention will describe now and use PBGS as the example of multimeric protein. Therefore, the invention provides a kind of composition, it comprises inhibitor, this inhibitor suppresses to have the formation of the polymer porphobilinogen synthase activity form of the first quantity monomer by being attached to the polymer porphobilinogen synzyme than the low activity form with second quantity monomer, wherein the first quantity of monomer is more than the second quantity.

The present invention finds that PBGS can be with the quaternary structure of at least two kinds of replacements, and eight aggressiveness and six aggressiveness exist. Polymer PBGS with lesser amt PBGS monomer is also included among the present invention. Before, only know existence eight aggressiveness forms. In two kinds of forms, monomer comprises and comprises 8 barrels of terminal 300 amino acid whose α 8 β of C, and wherein the central authorities of 8 barrels of α 8 β comprise avtive spot. The variable-length N end portion of this subunit forms the arm configuration that extends, and it is that subunit-subunit interaction is relevant widely in eight aggressiveness and six aggressiveness with two kinds of oligomer forms. The main difference of these two quaternary structure is configurations of the terminal arm of N.

In certain embodiments, polymer porphobilinogen synzyme supposes that from bacterium, archeobacteria or eucaryote eight aggressiveness porphobilinogen synzyme comprise the magnesium binding site of allosteric. In a variant of the present embodiment, polymer porphobilinogen synzyme comprises the zinc binding site of catalysis.

Following concept and definition are used for helping those skilled in the art to understand detailed description of the present invention.

The MORPHEINS concept:

The three-dimensional structure that a doctrine of modern biochemistry is protein is that the amino acid sequence of this protein directly determines. As a result, we are instructed the sequence of an albumen to determine its natural structure. The concept of a structure has been challenged in the discovery of prion, if but there is the people to think that is " false folding ", then he can adhere to above-mentioned doctrine. The present invention has drawn new discovery, i.e. morpheins, and it is albumen quaternary structure alternately, the result that the physiology that the configuration of monomeric unit changes is relevant. With regard to morpheins, the native state that replaces each other energy approaches, but the different limited quaternary structure diversity of each state regulation is schematically shown such as Figure 11-14. First example of morpheins is porphobilinogen synzyme (PBGS) system, and wherein the quaternary structure balance forms the architecture basics of mutamerism.

In Figure 11-14, infrastructure element is monomer, and the combination of any two unit is subject to the driving that contiguous thick line is placed dotted line. This is the rule of set. Figure 13 bidimensional ground shows that the diversity of set is subjected to the concept of the guidance of infrastructure element (being shown as monomer) shape and aggregation rules. Base unit has four kinds of difformities in Figure 13. The monomer pac-man-like shape in the lower left corner can not combine with the mode of himself placing dotted line with adjacent thick line, and this monomer can not oligomerization. Semiellipse monomer shape can with himself combine the formation dimer. In case the formation dimer, all dotted lines are adjacent with all thick lines, and oligomerization finishes with dimer. The pie wedge-shape can be in an identical manner and himself combination, and all dotted lines and all thick line are adjacent but shape needs three unit. The destiny of this pie wedge monomer is that oligomerization turns to tripolymer. At last, according to identical logic, the destiny of square individual form is to form tetramer set. Adopt the morphein concept, each monomer has the relevant functional character of different physiology, for example different Km and Vmax value. For example, polymer can be allosteric " open mode " has enzymatic activity high and other polymer can be " closed condition " of allosteric has low activity. Perhaps, the function of different oligomer can be the result of oligomer molecular surface difference. For example, trimerical circular surface will interact from acceptor and the binding partners different with tetrameric point surface with dimeric oval surface among Figure 11-14. The celluar localization of these molecular surface difference regulation compounds.

Allosteric is total concept, and wherein the activity of enzyme is subjected to the allosteric Molecular regulator to be attached to the impact of the binding site in on-catalytic site on the albumen. What the model suggestion that most of allosterics are regulated was active is identical multifarious oligomer with inactive state, and these two kinds of forms are in balance each other, and the combination of allosteric Molecular regulator can be moved this balance. Generally speaking, structural information (for example 3 D X-ray crystal structure) is not enough to understand the position of allosteric regulatory factor combination, and how two kinds of forms produce difference each other, and why a kind of form is higher than other form activity. Two kinds of different quaternary structure forms of our recent findings porphobilinogen synzyme (PBGS) wherein can check these forms and infer that some organisms are by the reasonable dismissal of magnesium allosteric adjusting PBGS. First object lesson that this discovery is morpheins also is described in detail among the application. Yet the observation of the quaternary structure that PBGS is replaced causes the morpheins concept as total description of the regulation mechanism of protein function with to catching one or another kind of morpheins form with the description of the reagent that instructs protein function. Figure 11 example use the tetramer and trimerical this conception of species for example shown in Figure 13, can only be attached to the tetramer but added sliver with explanation allosteric regulatory factor. The combination of sliver is disturbed the balance of quaternary structure and is made system towards tetramer form. Figure 14 illustrates the required quaternary structure state that can catch albumen and the concept that therefore balance is pulled to the reagent (being shown as wedge) of this state. Therefore, these will disturb the reagent of morpheins quaternary structure balance can suppress or activated protein. In one embodiment of the invention, this reagent is inhibitor, and it is caught inactive form and therefore stop and forms activity form. In one embodiment, this albumen is PBGS, and inactive form is six aggressiveness and activity form is eight aggressiveness. The nonrestrictive example of this inhibitor is the Rosmarinic acid or derivatives thereof. In another embodiment of the invention, reagent is activator, and it catches the activity form of this albumen.

Term " promoter " or " promoter region " refer to usually the nucleotide sequence found in coded sequence upstream (5 '), and its recognition site or start in correct position by RNA polymerase is provided is transcribed other essential factors and controlled the generation of mRNA (mRNA) and the expression of control coding sequence. Promoter described here or promoter region comprise by being connected to various regulating and controlling sequences, sudden change at random or control, and add or repeat enhancer sequence and the promoter of deriving changes. Promoter sequence disclosed herein, and the coded sequence that the biological function equivalent be responsible for to drive after a part that is used as suitable recombinant vector is incorporated among the host under its control transcribes, and embodies as its ability that produces mRNA.

" regeneration " refers to from the process of plant cell (for example plant protoplast or explant) growing plant.

" conversion " points to the method for introducing exogenous nucleic acid sequences (for example, carrier, recombinant nucleic acid molecules) in cell or the protoplast, and wherein exogenous nucleic acid is integrated in the chromosome or can self-replicating.

" transformant " is the cell that has changed DNA by introducing exogenous nucleic acid molecule in the cell.

Term " gene " refers to chromosomal DNA, DNA, cDNA, synthetic DNA, or the DNA of other encoded peptides, polypeptide, albumen, or the both wings zone of RNA molecule and the coded sequence relevant with expression regulation.

Phrase " to the dna fragmentation of promoter region allos " presentation code DNA section is not natural be present in at present with homologous genes that promoter is connected in.

Term " coding DNA " refer to the to encode chromosomal DNA of any enzyme discussed herein, DNA, cDNA, or synthetic DNA.

Term " genome " had both comprised that the chromosome in the bacterial host cell also comprised plasmid when it is used for bacterium. Be incorporated in the bacterial host cell coding DNA of the present invention therefore or chromosomal integration or be positioned in the plasmid. Term " genome " not only is included in when it is applied to plant cell in the chromosomal DNA of finding in the nucleus, also is included in the organelle DNA of finding in the subcellular components of cell. The present invention be incorporated in the plant cell DNA therefore or chromosomal integration or be positioned in the plasmid.

Term " herbicide " refers to be used to the chemical substance of killing or suppress plant, plant cell, vegetable seeds or plant tissue growth.

Term " inhibitor " refers to for example chemical substance of the enzymatic activity of crucial biosynthetic enzyme, acceptor, signal transducer, structural gene product and transport protein concerning the growth of organism or existence of deactivation albumen. In the context of the present invention, inhibitor is the chemical substance of the enzymatic activity of deactivation porphobilinogen synzyme. Term " herbicide " is used for defining the inhibitor that is applied to plant, plant cell, vegetable seeds or plant tissue herein.

Term " microorganism " or " microbial body " refer to algae, bacterium, archeobacteria, fungi and protozoan.

" overexpression " refers to by the polypeptide of the dna encoding that is incorporated into host cell or protein expression, wherein said polypeptide or albumen or be present in the host cell abnormally, or described polypeptide or albumen are to be present in the described host cell than usually expressing higher level by the endogenous gene of coding said polypeptide or albumen.

Term " plant " refers to be in the part of plant or the plant of any stage of development. Also comprise cutting herein, cell or tissue culture and seed. The term that uses among the present invention " plant tissue " includes but not limited to that whole plant, plant cell, plant organ, vegetable seeds, protoplast, callus, cell culture and tissue form any colony of the plant cell of structure and/or functional unit.

Term " plastid " refers to comprise amyloplast, chloroplaset, chromoplast, oleoplast, eoplasts, etioplast, the plant cell organelle kind of leucoplast and protoplast. These organelles are self-replicating degree and the ring-shaped DNA molecule that comprises so-called " chloroplast gene group ", and its magnitude range depends on floristics from about 120kb to about 217kb, and usually comprise the inverted repeat zone.

Term " solid " refers to the non-aqueous component of stem tuber (in potato) or fruit (in tomato), mainly comprises starch and other polysaccharide, simple sugars, non-structural carbohydrate class, amino acid and other organic molecules.

Term " tolerance/opposing " refers to when being exposed to inhibitor or herbicide after continuation normal growth or the ability that works.

Term " conversion " points to the process of introducing allogeneic dna sequence DNA in cell, tissue or the plant. The cell that transforms, tissue or plant are understood to not only comprise the end-product of conversion process, also comprise its transgenic progeny.

" oral composition " is a kind of product, it is swallowed intentionally for the purpose of the concrete therapeutic agent of whole body administration in common use procedure, but is retained in the oral cavity one sufficiently long period with basic contact all dental surface and/or oral cavity tissue for the reason of orally active. Oral composition can be that single-phase oral composition maybe can be the combination of two or more oral compositions.

The medium that term used herein " oral acceptable carrier " expression is suitable, it can be used to use the present composition to the oral cavity with safety and effective mode. The material that such medium comprises is fluoride sources, other antilithic for example, buffer, other grinding-material, peroxide source, alkali metal hydrogencarbonate, thickener, NMF, water, surfactant, titanium dioxide, spices system, sweetener, xylitol, colouring agent and composition thereof.

Term " morpheins " is used to describe the different quaternary structure isoforms of the albumen with difference in functionality characteristic (for example, different catalytic property) in the present invention is open. The other term that is used for these quaternary structure isoforms comprises " quatreins ", " isoquatomers " and " selkeins ". Selecting the term of back is according to the selkie in the mythology, and it can change form corresponding to special stimulation and function. With regard to plant and some bacterium PBGS, the stimulation of changing between morpheins is the allosteric regulatory factor, that is, reagent (as, magnesium).

The Morpheins of given albumen has part difference at secondary and tertiary structure, and these differences have determined the difference of quaternary structure. In some aspects, the similar PrPC of morpheins; The sequence of an albumen can experience configuration and change, and it produces the variation (coherent condition) of quaternary structure. Yet aspect other, the difference of morpheins and PrPC is that the change that oligomer belongs to fixing diversity and quaternary structure is irreversible, non-pathogenic, and be the part of normal physiological regulation process.

Therefore, in the present invention, total mechanism that suggestion uses the allosteric of morpheins (quaternary structure isoform) to regulate. In this mechanism, monomer structure is different from some aspect of their secondary/tertiary structure, and these differences have determined that set becomes one or another kind of morphein. This mechanism is illustrated schematically among Figure 11-14.

Figure 11 is that the bidimensional of the balance between two kinds of forms of albumen (morpheins) represents. A kind of unit of form (such as, monomer) (being shown as square herein) contains four kinds of different surfaces, is line, thick line, dotted line and meander line. Naturally the complementary surface that links to each other is described as thick line and dotted line at this. This contact defines the rule that engages between the unit. When the subunit of square contact potential is satisfied (in other words when all thick lines and all dotted line contact), the optimized set that obtains is the tetramer. Therefore, the oligomer set is by the structure of monomer and the rule predetermining of joint. Shown in Figure 11, box structure and " sliver (splinter) " contact, it is the graphic form of reagent (for example, allosteric regulatory factor molecule); Square monomer and the square tetramer and sliver are contacted the function that affects multimeric protein, and for example with regard to plant and some bacterium PBGS, magnesium provides the stability of these albumen forms.

Square unit and another structure are in balance, and the latter has some right rather than whole secondarys identical with tertiary structure with the former, therefore only total surfaces characteristic.Alternative unit is shown in the section among Figure 11.This monomer contains the surface of being described by thick line and dotted line; The rule that engages between these surfaces is regular identical with rectangular cells.As a result, follow this joint rule, alternative unit assembling becoming trimer.The binding site that trimer structure and its independent component do not contain allosteric regulatory factor molecule (sliver) is important.Because sliver is stablized square and oligomer thereof, exist sliver to pull to square and its oligomer to the balance of quarternary structure.

Observation to six aggressiveness PBGS provides quarternary structure how to be used as first example that allosteric is regulated the architecture basics of protein function.In the PBGS from photosynthetic organism and some antibacterials, the protein concentration dependency of specific activity provides the equilibrated evidence (referring to Fig. 5 A) between full activity (supposition is eight aggressiveness) form and deactivation (supposition the is six aggressiveness) form.

Figure 11 is the description overall to the PBGS behavior.With regard to PBGS, square is to embrace dimer and section is a dimer separately.Under each situation, certain structures is identical, but not every surface character is identical, and the joint rule between the surface must be a first approximation total between two alternative structures.With regard to PBGS, the difference of oligomer structure changes the difference of functional characteristic into.Can suppose reasonably that other proteic different quarternary structure also change the difference of functional characteristic into.The dimerization of known receptor is relevant with signal transduction.Still unrecognizedly before the present invention be, in the dimeric structure monomeric structure can with when this monomeric structure is not in dimeric structure, be different.

Another the nonrestrictive example of albumen that contains morpheins is an Ia type ribonucleotide reductase.The updated model that regulate to propose for Ia type ribonucleotide reductase allosteric describe balance between the tetramer and six aggressiveness (Cooperman and Kashlan, 2003, Adv.In EnzymeRegulation, 43:167-187).In this example, the purpose that model is for the purpose of illustration only and the author does not have the difference that protein structure defines the morpheins of supposition.Yet ribonucleotide reductase is crucial for the from the beginning biosynthesis of DNA, and Ia type enzyme is found in all eukaryotes, and the from the beginning biosynthesis of inhibition DNA is the reasonable approach of cancer chemotherapeutic.Therefore can be by optionally effector being attached to function (for example, suppressing) to realize influencing Ia type ribonucleotide reductase than the surface of low activity morphein uniqueness.

Multimeric protein of the present invention should have at least one characteristic, the specific activity that relies on of protein concentration or for example by for example ion exchange, native gel electrophoresis, analytical ultracentrifugation, size exclusion chromatography (according to size) and be separated into the ability of different set.

For proving above-mentioned balance, can carry out dynamics research to show for example K _mAnd V _MaxBe suitable for the activity as the concentration of substrate function of MM equation; Morpheins is hyperbola of fit but match curve (referring to Nature Structure Biology) in pairs well.

The example of the unrestricted type of multimeric protein function be enzymatic activity and with the ability of other interactions of molecules, for example, in conjunction with different proteic abilities.This function can be suppressed or strengthen.The change of monitoring function can be by for example monitoring the kinetic parameter K that those skilled in the art understand _mAnd V _MaxCarry out.In certain embodiments of the invention, come the function of Profilin by stable more SA morphein.

In certain embodiments, the function of agents influence multimeric protein.The nonrestrictive example of the function of multimeric protein is active, wherein influence be suppress or activation at least a.In certain embodiments, reagent is with to have more SA quarternary structure isoform relevant.In certain embodiments, reagent is attached to the quarternary structure isoform with greater activity.The nonrestrictive example of the reagent that suppresses eight aggressiveness PBGS is hereinafter described.

The eight aggressiveness forms of PBGS are attached to substrate with the relevant concentration range of physiology, and are activated under physiological pH.Eight aggressiveness are embraced dimer by four and are formed, and the arm of one of them subunit interacts to bucket with strong bucket and holds the barrel-like structure of adjacent subunit tightly.

Newfound eight aggressiveness form PBGS are biosynthetic key components of regulation and control tetrapyrrole in the subclass of organism, and this subclass comprises plant and some malignant bacterias, but does not comprise the people, animal and fungus.Six aggressiveness forms are non-activity substantially under physiological condition.Particularly, six aggressiveness can not be in physiology related concentrations scope bound substrates because its K _mValue is than the K of eight aggressiveness _mValue is at least two orders of magnitude greatly.Six aggressiveness are made up of three dimers that separate, wherein the N-terminal arm not with adjacent subunit with strong bucket to the bucket contact interaction.

Conversion between six aggressiveness and the eight aggressiveness forms comprises the remarkable change of protein structure.Referring to, as Fig. 5 A.Certain embodiments of the present invention relate to the structural change of inhibition from six aggressiveness to eight aggressiveness, with the activation of PBGS and the biosynthesis of tetrapyrrole in inhibition plant and/or the antibacterial.Because it is effective to plant and antibacterial to suppress mechanism, and invalid to animal, the invention provides and suppress antibacterial, antibiotic and herbicide new approach in application.

Therefore, in certain embodiments, the present invention includes the inhibitor that those PBGS are changed to eight aggressiveness from six aggressiveness by magnesium regulation and control back under physiological condition.Inhibitor can be known or new chemical compound.Inhibitor significantly carries out six aggressiveness effectively suppress tetrapyrrole in the conversion of eight aggressiveness biosynthesis on the physiology in the growth of plant and bacterial pathogens and propagation process.To the inhibition of changing to the quarternary structure of eight aggressiveness PBGS from six aggressiveness PBGS is the new target of exploitation antibiotic and herbicide.

Some variations are arranged in the proteic system of the PBGS generating process, and some of them albumen has the magnesium of allosteric, and other albumen do not have.PBGS with magnesium of allosteric comprises the archeobacteria territory, all antibacterials except that red antibacterial (Rhodobacter) belongs to and all photosynthetic eukaryote territories (as green plants) (24).Another exception is malarial parasite plasmodium (Plasmodiumfalciparum) recently.The structure of the crystal structure of escherichia coli (E.coil) PBGS that had before determined according to the inventor and six aggressiveness PBGS disclosed herein it seems that the effect of magnesium of allosteric is the structure conversion of inducing between low activity six aggressiveness and high activity eight aggressiveness.Six aggressiveness-eight aggressiveness the conversion that Mg acts on the PBGS is the new structure example that allosteric is regulated protein function.

The structure of E.coli PBGS is shown in Fig. 9 A-C and is used for showing the melts combine variation that the PBGS structure is common.Each E.coli PBGS monomer contains two metal ions, does not guard on system takes place for these two.Avtive spot contains the active crucial zinc ion to E.coli PBGS, but its three cysteine parts do not exist in many PBGS.Zinc works in the combination of second substrate molecule (33) and reactivity.The details in zinc site is shown in Fig. 9 B.In addition, the visible N-terminal arm with adjacent subunit of the magnesium of allosteric is at each α, the combination at the interface of β bucket, and CONSTRUCTED SPECIFICATION is shown in Fig. 9 C.Sequence-dependent determinant in conjunction with the magnesium of allosteric is not present among all PBGS.

Fig. 6 shows whether use avtive spot zinc and whether use the magnesium of allosteric that it is divided into the sketch map of four classes (24) according to PBGS.First matrix (the most left) is divided into two classes: (a) the avtive spot zinc (shade) on the left side and (b) the non-activity site zinc (shadow-free) on the right.Second matrix is divided into two classes: (a) magnesium of the no allosteric at top (rhombus) and (b) magnesium (square) of the allosteric of bottom.Make up two matrixes the matrix of being made up of four quadrants (the rightest) is provided, Northwest Quadrant (QNW) representative+Zn/-Mg wherein, Northeast Quadrant (QNE) representative-Zn/-Mg, Southwest Quadrant (QSW) representative+Zn/+Mg, southeast quadrant (QSE) representative-Zn/+Mg.

The inventor the is quantitative in the past following distribution of (24) known array to four quadrants: QNW=9; QNE=2; QSW=55 and QSE=63.Therefore, the present avtive spot zinc that needs of the only about half of coding of available sequences, and half is opposite in addition (that is QNW+QSW～QNE+QSE).Opposite with the distribution of avtive spot metal mode, surpass the bonded determinant of magnesium that 90% PBGS sequence contains allosteric (that is, QSW+QSE＞＞QNW+QNE).

The PBGS of avtive spot zinc of subgroup (that is the PBGS in the QSE) inhibitor does not contain to(for) the magnesium that contains allosteric is the most effective.These subgroups are subgroups of photosynthetic eukaryote and antibacterial, comprise for example Pseudomonas aeruginosa of pathogen.These PBGS albumen cause the specific activity character that protein concentrations rely on, and it has indicated the mutual conversion between active bigger quarternary structure form and the active less quarternary structure form.With reference to the NE quadrant, wherein primary evidence shows that activity form is six aggressiveness in addition.

Therefore, in certain preferred aspects, inhibitor of the present invention effectively suppresses the formation from antibacterial, archeobacteria or Eukaryotic eight aggressiveness PGBS, supposes that eight aggressiveness PGBS contain the magnesium binding site of allosteric.The nonrestrictive tabulation in the eight aggressiveness PGBS sources that can be suppressed by compositions of the present invention is shown in Fig. 8, and it is the classification that comprises the organism in antibacterial territory, archeobacteria territory and eukaryote territory.But Fig. 7 A and 7B are represented in April, 2002 from GenBank and the comparison of the avtive spot melts combine residue of the PBGS sequence that the genome of other network retrievals obtains.The information that shows based on Fig. 7 is assigned to organism in one of four quadrants of Fig. 6.The clump (people PBGS the 122nd, 124 and 132) of being rich in cysteine is in conjunction with being positioned at the existence that arginine residues (the 221st of people PBGS) on the avtive spot lid (lid) shows avtive spot zinc binding site.Do not have and be rich in cysteine site zinc to contain the zone and the avtive spot lid residue that are rich in aspartic acid on the contrary in conjunction with the kind of clump be lysine.

In certain embodiments of the invention, thereby inhibitor substituted metal ion is combined in metal ion binding site, and the preferable alloy ion is zinc or magnesium.In certain embodiments of the invention, inhibitor is combined in avtive spot.Inhibitor can be combined in Anywhere, but binding site must be stablized a kind of quarternary structure.Preferred combination is to the site that does not exist in another kind of polymer at a kind of polymer.

Inhibitor of the present invention can use following scheme to identify.At first, provide the magnesium that comprises allosteric and the model that does not comprise six aggressiveness form PBGS of avtive spot zinc.Initial model can be one of Semen Pisi sativi PBGS for example.Secondly, computer virtual screening micromolecule data base can be adapted to the molecule of the domain that can not implement embrace adjacent with the N-terminal part of subunit with searching.The domain that can not implement to embrace is dimeric at least one zone separately, and inhibitor is attached to and suppresses dimeric arm on this zone and hold dimeric bucket tightly, and this is that to form another dimer necessary to form active eight aggressiveness.Referring to Figure 10, wherein circle is represented inhibitor.The possible site of the domain that can not implement to embrace is positioned under the junction (that is, being positioned at " axillary fossa " locates) of holding arm binding subunit main body tightly.Suitable in theory molecule will be measured by experience external by the effect of measuring the specific activity that they rely on the protein concentration of Semen Pisi sativi PBGS, and this can use artificial gene construct to obtain.These molecules with protein concentration mode Profilin specific activity are good candidate inhibitors.

Following method will allow to identify the inhibitor that is attached to Anywhere, must not form to suppress eight aggressiveness by the domain that can not implement to embrace on PBGS.The functional analysis of PBGS specific activity will at first be used for selecting potential inhibitor, the material that described molecule is for example harmless to the people from the available molecule that the computer screening is identified.After having selected potential inhibitor, they will further screen the active influence of contrast according to protein concentration.

Therefore, the invention provides the method that influences multimeric protein, this method comprises: provide to comprise the multimeric protein with a plurality of unit sets, wherein each described unit comprises that first complementary surface is relevant with another unitary second complementary surface with second complementary surface and one of them unitary first complementary surface, suppose that this set is at least a in the different quarternary structure isoforms, condition is the structure of the described different quarternary structure isoform of (1) described unitary structures shape, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quarternary structure isoforms; The compositions of the present invention that comprises reagent is provided, wherein this reagent influences balance by the binding site that is attached in this set, and should gather with reagent and contact, wherein this reagent influences balance by being attached to binding site and influencing described multimeric protein thus.In some embodiment of this method, influence the formation that described multimeric protein comprises influences the quarternary structure isoform.In some embodiment of this method, influence the function that described multimeric protein comprises influences described multimeric protein.

Also provide and suppress the method that polymer porphobilinogen synzyme forms activity form, this method comprises: compositions of the present invention is applied to polymer porphobilinogen synzyme; With compositions with get in touch than the low activity form; Thereby inhibition forms activity form than the assembling of low activity form and suppresses polymer porphobilinogen synzyme and forms activity form.The nonrestrictive example of inhibitor is the rosmarinic acid or derivatives thereof.

It is growth or the growth that is used for suppressing or preventing human or animal host antibacterial territory, archeobacteria territory and/or eukaryote territory that present composition thing is preferably used.Other application of the present composition comprise prevention or suppress the biomembrane that various surfaces comprise tooth, pipeline, steamer or are immersed in any surface in water/air mixture, and the antibacterial that loses may be found to cause in above-mentioned various surfaces.Therefore, for example, the growth of barnacle on the steamer surface can effectively be prevented or suppress to compositions of the present invention.

The organism that depends on targeting, compositions of the present invention can be used to the loss that prevents or prevent to be caused by some kind.The example of the organism among the QSE is at table 1, so these organisms are main targets of using compositions of the present invention.Use table 1 as guide, the various application of the present composition it is contemplated that into, for example medicine, toothpaste, soap, disinfectant, antibiont film composition and herbicide.

Table 1 has the antibacterial of the PBGS of SE quadrant

The species title	Possible loss
		Yersinia enterocolitica (Yersinia enterocolitica)	The gastroenteritis that food causes
Yersinia pestis (Yersinia pestis)	The plague
		Pseudomonas syringae (Pseudomonas syringae)	Phytopathogen (Rhizoma Solani tuber osi)
Pseudomonas aeruginosa (Pseudomonas aeruginosa)	The facultative pathogen of people's wounded tissue is with to antibiotic resistance and well-known
		Actinobacillus actinomycetem comitans (Actinobacillus actinomycetemcomitans)	Periodontal
Pasteurella multocida (Pasturella multocida)	Import the factor (invective factor) in the animal bite
		Corrupt Shiva bacterium (Shewenella putrefasciens)	Corrosion of Oil Pipeline, fish is addled
Pod membrane methyl coccus (Methylococcus capsulatus)	Use methane as carbon source
		Vibrio cholera (Vibrio cholerae)	Cholera-serious diarrhoea
Xyllela fastidiosa (Xylella fastidiosa)	Pierce's disease in the plant (as, Fructus Vitis viniferae)
		Crescent handle bacillus (Caulobacter crescentus)	Asymmetric cell division
Agrobacterium tumefaciems (Agrobacterium tumefaciens)	Flos Rosae Rugosae and other are as root knots such as Fructus Mali pumilae, pears, peach, Fructus Pruni pseudocerasis
		Sinorhizobium meliloti (Sinorhizobium meliloti)	The nitrogen fixing bacteria of alfalfa
Malta's brucella (Brucella melitensis)	The bacterial disease of domestic animal (sheep, goat).People's Malta fever.
		Rhodopseudomonas palustris (Rhodopseudomonas palustris)	Purple Non-sulfur phototropism antibacterial
Mesorhizobium loti	The object of biotechnology

The living slowly root nodule bacteria of Semen sojae atricolor (Bradyrhizobium japonicum)	The fixed nitrogen Semen sojae atricolor
		Brucella melitensis biovar (Brucella melitensis biovarsvis)	The beastly disease that passes mutually of Prussia's bacterium disease-people
Magnetospirillum magnetotacticum	Form magnetic iron ore
		Inner Mongol rickettsia (Rickettsia conorii)	Mediterranean spotted fever
Rickettsia prowazekii (Rickettsia prowazekii)	Epidemic typhus
		Novosphingobium aromaticivorans	Food industry
The special bacterium (Bordetella bromchseptica) of bronchitis Boulder	Be common in cat
		The special bacterium (Bordetella pertussis) of pertussis Boulder	Pertussis
Nitrosomonas europaea (Nitrosomonas europaea)	The auxotroph nitrobacteria
		Glanders burkholderia (Burkholderia mallei)	Glanders (horse); Possible bio-terrorism reagent
Melioidosis burkholderia (Burkholderia pseudomallei)	Melioidosis, WhitmoreShi disease, tropical climate's endemic diseases
		Burkholderia fungorum	" group ", pathogen of people and plant and the antibacterial important to environment
Neisseria meningitidis (Neisseria meningitides)	The antibacterial meningitis
		Neisseria gonorrhoeae (Neisseria gonorrhoeae)	Gonorrhea
Ralstonia solanaccarum	Plant disease, " southern wilt disease (Southern wilt) "
		Ralstonia metallidurans	Preventing from heavy metal
Chlamydia muridarum	Chlamydia-STD
		Sand holes chlamydia (Chlamydia trachomatis)	Chlamydia-STD
Chlamydia pneumoniae (Chlamydophila pneumoniae)	10% of pneumonia
		Chlamydia psittaci (Chlamydophila psittaci)	Psittacosis
Chlorobium vibrioforme	Green sulfur bacteria
		Clorobium tepidum	Green sulfur bacteria
Rhodothermus marinus	The antibacterial of thermophilic acidophilic salt
		Cytophaga hutchinsonii	The digestion crystalline cellulose
Shewanella oneidensis	Soluble metal ion can be transferred to insoluble, bioremediation
		Vibrio vulnificus (Vibrio vulnificus)	The temperature sea water infects open wound
Vibrio parahaemolytious (Vibrio parahaemolyticus)	The temperature sea water infects open wound; Diarrhoea
		The yellow sporangium (Xanthomonas campestris) in field	Phytopathogen
The yellow sporangium (Xanthomonas axonopodis) of carpet wool	Phytopathogen
		Little pyriform Pseudomonas (Pirellula)	Phytopathogen
Brown algae Fucus Vesiculosus (Brown algae Fucus vesiculosus)	Fucus Vesiculosus

Advantageously, the disease that compositions of the present invention is effectively treated or prevention is caused by antibacterial, archeobacteria and/or eukaryote.Said composition effectively prevents to form polymer PBGS (as, eight aggressiveness PBGS or have monomeric other activity forms of lesser amt) thereby and suppresses or prevent antibacterial, archeobacteria and/or Eukaryotic growth or growth.In certain embodiments, polymer PBGS contains the magnesium binding site of allosteric.In a variant of this embodiment, the disease that said composition is effectively treated or prevented to cause owing to contact antibacterial, archeobacteria and/or eukaryote.In another variant of this embodiment, said composition is at least a in medicine, toothpaste, soap, disinfectant, antibiont film composition and the herbicide.

In certain embodiments, said composition does not comprise the magnesium binding site and the catalytic zinc of allosteric.In a variant of this embodiment, the disease that said composition is effectively treated or prevented to cause owing to contact antibacterial, archeobacteria and/or eukaryote.In another variant of this embodiment, said composition is at least a in medicine, toothpaste, soap, the disinfectant.

Antibiotic, herbicide and antifungal usually based on to antibacterial, plant or fungus special and be not present in the inhibition of the critical path in people/animal.For example, 1) biosynthesis of Penicillin antibiotics directed toward bacteria cell wall, and zooblast does not have cell wall, or 2) herbicide glyphosate is at the ArAA biosynthesis, and the people does not have this approach, we must eat ArAA.Along with we understand manyly more to the difference of the sequence of different albumen/enzymes and structure, possible more targeting is prevalent in the critical path in animal, plant, antibacterial, the fungus.The situation that targeting tetrapyrrole biosynthesis pathway comes to this as the basis of antimicrobial or herbicide by suppressing PBGS.The variation in various biological PBGS melts combine site provides enough architectural differences for the inhibitor that exploitation does not suppress people PBGS in system takes place.With regard to PBGS, PBGS the morphein form and between the aminoacid sequence on morphein surface equilibrated capability, different biologies have significant difference.With regard to stablize a kind of morphein form by selectivity more at large for the Profilin function, possible situation is, target is the approach that is not present in philtrum, and perhaps possible situation is, target only has enough systems and morphs outside avtive spot, because of the surperficial difference of morpheins very big.

In certain embodiments, said composition comprises the medicine acceptable medium except reagent.The medium of " medicine acceptable medium " expression for example can be sent in inhibitor and the compositions other any active agents to the solvent of target organism with comparatively safe and effective and efficient manner.This medium itself does not need to have any pharmaceutically active.

" medicine acceptable medium " used herein comprises any He all solvents, disperse medium, coating, antibacterium and antifungal, isotonic agent and delayed absorption agent etc.It is well known in the art that this medium and reagent are used for pharmaceutically active substance.Except unmatched conventional media of inhibitor any and of the present invention known today or reagent, all can consider the application in pharmaceutical composition.Supplementary active ingredients also can be incorporated in the compositions.

As the solution of the active component of free alkali or drug acceptable salt can by in water with surfactant, for example hydroxypropyl cellulose mixes suitably and prepares.Disperse system can also be at glycerol, liquid macrogol, more than both mixture and in oil, prepare.Under common storage and service condition, these goods comprise the antiseptic that prevents all growth of microorganism.

Compositions of the present invention can comprise traditional pharmaceutical preparation.The administration of the present composition will be via any common approach, as long as target tissue can be obtainable by this approach.Approach comprises mouth, nose, cheek, rectum, vagina or part.In addition, administration can be passed through original position, Intradermal, subcutaneous, intramuscular, endoperitoneal or intravenous injection.Will be under these compositions normal conditions with the administration of said medicine acceptable composition.

Compositions of the present invention also can prepare the solid that is suitable for making solution or suspension before injecting in liquid with the advantageously administration of form of the Injectable composition of liquid solution or suspension.These goods also can be emulsified.The exemplary composition that is used for this purpose comprises 50mg by every ml phosphate buffer or until the human serum albumin of about 100mg.The other drug acceptable carrier comprises aqueous solution, and non-toxic excipients comprises salt, antiseptic, buffer agent etc.The example of nonaqueous solvent is propylene glycol, Polyethylene Glycol, vegetable oil and injectable organic ester, for example ethyl oleate.Aqueous phase carriers comprises water, alcohol/aqueous solution, saline solution, and parenteral media is sodium chloride, RingerShi glucose etc. for example.The intravenous media comprises liquid and nutritional supplement.Antiseptic comprises antimicrobial, antioxidant, chelating agen and noble gas.The pH of different component and actual concentrations are according to known parameter adjustment in the medicine.

Other goods are suitable for oral administration.Oral products comprises typical excipient, for example the mannitol of pharmaceutical grade, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.Compositions is taked solution, suspension, tablet, pill, capsule, slow releasing agent or form of powder.When route of administration is a local time, medicament forms can be emulsifiable paste, ointment, ointment or spray.

The effective dose of therapeutic agent target on the estimation decides.Term " unit dose " refers to the suitable physically discontinuous unit that uses to object, and constituent parts comprises the pharmaceutical composition of scheduled volume, and this amount is calculated with the generation required reaction relevant with its administration, and described administration is suitable way and therapeutic scheme.Treat the amount of administration, number of times and unit dose according to treatment depend on the state of object to be treated, object and required protection.The accurate amount of pharmaceutical composition also depends on medical practitioner's judgement and is unique to each individuality.

The Another Application of the present composition is a herbicide, and wherein said composition comprises the medium of effective weeding in addition." the effectively medium of weeding " represents medium, for example can send the solvent of other any active agents in inhibitor and the compositions to the target organism.This medium itself does not need to have any activity of weeding.

Guide to the crop applying antibacterial compositions provides as follows: because all light compositing eukaryotes all fall into the QSE quadrant of Fig. 6, they itself are the targets of inhibitor of the present invention.Yet the axillary fossa inhibitor binding site that Figure 10 shows has significant phylogenetic difference between plant and antibacterial.

Therefore, must be that those are attached on this site of antibacterial PBGS as the reagent of crop antibacterium spray, and be not joined to the reagent in plant PBGS site.

Compositions of the present invention comprises diluted composition that preparation uses immediately and the needs concentrate composition of common dilute with water before use.

Solid composite can be a granule, and form of powder, wherein active component and ground solid diluent, for example Kaolin, bentonite, kieselguhr, dolomite, calcium carbonate, Muscovitum, magnesia powder, FullerShi soil or Gypsum Fibrosum or its combination mixing mutually.They can also adopt dispersible powder or particulate form, comprise that wetting agent is to promote powder or the dispersion of granule in liquid.The solid composite of powder type can be used as dust and uses.

Fluid composition can comprise solution, suspension and the disperse system of active component in water, and described water is optional to contain surfactant, maybe can comprise solution or the disperse system of active component in the mixable organic solvent of water, and it is dispersed in the water with droplet.Herbicidal composition is adapted at mixing in the jar with preparation and prepares the diluted composition that uses immediately or be used to form concentrate.

The preparation method of solution or disperse system is that solubilization of active ingredient is comprised in the water or organic solvent of wetting agent or dispersant optional, when with an organic solvent, the mixture that as above obtains is added to optional comprising in the water of wetting agent or dispersant then.Suitablely comprise when organic solvent, for example, dichloroethylene, isopropyl alcohol, propylene glycol, diacetone alcohol, toluene, kerosene, methyl naphthalene, dimethylbenzene or trichloroethylene or its combination.

Other additive and adjuvant also can be present in the compositions of the present invention.Example comprises antifreeze for example ethylene glycol and propylene glycol; Dyestuff; Dispersant; Rheological agent; Antifoam is for example based on the reagent of silicone; With wetting agent ethylene glycol for example.

The exploitation of herbicide allows exploitation Herbicid resistant crop on this basis, its method is to make these resistance crops to the PBGS in two quadrants in top in four quadrants of Fig. 6, for example people PBGS is transgenic (that is, comprising from the artificial hereditary material that shifts of other species).

Herbicide resistant plants

Also provide polymer porphobilinogen synzyme genetically modified herbicide resistant plants, described enzyme exists to embrace dimeric polymer form substantially.In certain embodiments, polymer porphobilinogen synzyme is from the people.In certain embodiments, polymer porphobilinogen synzyme does not contain the magnesium binding site of allosteric.Provide preparation to be adapted to the guide of the genetically modified herbicide resistant plants of polymer porphobilinogen synzyme below.

The expression of gene in plant that exists with the double-stranded DNA form comprise messenger RNA (mRNA) from the chain of DNA by rna polymerase transcribe and subsequently the mRNA primary transcribe in endonuclear processing.This processing relates to 3 ' untranslated region, adds polyadenylic acid at the 3 ' end of RNA.DNA is transcribed into the adjusting that RNA is subjected to the DNA zone of so-called promoter.Promoter region comprises and signals to RNA polymerase, it is connected and a chain using DNA starts that mRNA transcribes and the base sequence that produces corresponding mRNA complementary strand as template with DNA.This mRNA is used as template then, produces wherein encoded protein by the cell biological synthesis machine.

In the present invention, the promoter of selection should have the required tissue and the specificity of growth.Therefore, promoter function should be by selecting to have the approaching promoter of required tissue expression ability and tripping force and selecting to produce the required active transformant of PBGS and optimised.Usually adopt the allos expression of structural gene of plant from transforming the subpool method for screening, this is to cause there are differences (being commonly referred to " position effect ") containing between the transformant of identical heterologous gene because the gene in the Plant Genome inserts the site.Cause the promoter that DNA transcribes in the plant cell (composing type or organizing specific) except known, other promoter can be separated promoter region and be used for the present invention by evaluation by methods known in the art then by screen the gene that the plant cDNA library is searched optionally or preferably expressed in the required time.

In the preferred embodiment of the invention, the PBGS transgenic is expressed response light in chloroplast.More specifically, the PBGS transgenic is transcribed into mRNA in nucleus, and mRNA is translated into precursor polypeptide (chloroplast transit peptides (CTP)/PBGS) in Cytoplasm.The precursor polypeptide is equipped with transhipment (introducing) then in chloroplast.Active several chloroplasts of tool can photoinduced promoter be described in the literature in plant cell.The example of such promoter comprises from profuse plant polypeptide ribulose-1,5-bisphosphate, the small subunit of 5-diphosphonic acid carboxylase (ssRUBISCO) can photoinduced promoter, chlorophyll a/b binding protein gene promoter and be used to the phytochrome promoter (Shimizu-Sato etc., 2002) of light controllable initiating subsystem recently.Some have been used to be created in various types of DNA construct of expressing in the plant in these promoteres; Referring to the open WO 84/02913 of for example PCT.

Other are known or find that the promoter that causes DNA to transcribe in response to light in plant cell can be used for the present invention.Such promoter can obtain from various sources, for example plant and plant virus, include but not limited to enhanced CaMV35S promoter and from plant gene ribulose-1,5-bisphosphate for example, the promoter that the Gene segregation of the small subunit of 5-diphosphonic acid carboxylase (ssRUBISCO) arrives.As mentioned below, the preferred concrete promoter of selecting should be able to cause expressing fully produces enough tetrapyrroles with the PBGS enzyme that produces effective dose and keeps growth.In one embodiment, described promoter be seepage so that the necessary tetrapyrrole of non-light compositing function in the plant to be provided.

The expression that the active plastid of PBGS instructs

In the preferred embodiment of the invention, the PBGS gene fusion to CTP, with the PBGS targeting proteins to plastid.To comprise various forms of plastids as chloroplast and the plastid that hereinafter uses, comprise amyloplast.Many albumen that are positioned plastid are expressed as precursor and are targeted to plastid by CTP from karyogene, and CTP is removed in introducing step subsequently.The example of such chloroplast protein comprises ribulose-1,5-bisphosphate, 5-diphosphonic acid carboxylase (ssRUBISCO, SSU) small subunit, 5-enol acetone acid shikimic acid-3-phosphate synthase (EPSPS), ferredoxin, ferredoxin oxide-reductase is caught photoreactivation body protein I and protein I I and thioredoxin F.The epsp synthase plant gene of glyphosate tolerant is also encoded and is contained the polypeptide of CTP, and it makes the epsp synthase polypeptide be entered chloroplast (U.S. patent 5310667) in the plant cell by transhipment.Having shown that non-plastid albumen can merge by the albumen that use has a CTP is targeted to chloroplast, and the CTP sequence is enough to targeting proteins to plastid.Those skilled in the art will recognize that also can produce the function of utilizing concrete plastid transit peptides is incorporated into various other chimeric construct bodies in the plant cell plastid with the PBGS enzyme.The PBGS gene can also be by being targeted to plastid (Daniell etc., 1998) with gene transformation to the chloroplast gene group.In general, the chloroplast absorption signal such as CTP is rich in Ser, Thr and little hydrophobic amino acid residue.

The RNA that DNA construct of the present invention produces can also comprise 5 ' untranslated homing sequence.This sequence can be used for expressing gene ground promoter and can be modified to strengthen the translation of mRNA by specificity derived from selecting.5 ' untranslated region can also be from viral RNA s, from suitable eukaryotic gene, or obtains from synthetic gene order.The invention is not restricted to the construct of untranslated region from the 5 ' untranslated region that is accompanied by promoter sequence.On the contrary, the homing sequence of untranslated can be from incoherent promoter or coded sequence.

In monocotyledon, preferably in gene construct, comprise the expression that intron was beneficial to or strengthened coded sequence.The example of suitable intron comprises HSP70 intron and rice actin intron, and the both is well known in the art.Another suitable intron is Semen Ricini catalase intron (Suzuki etc., 1994).

The polyadenylic acid signal

3 ' untranslated region of chimeric plant gene is included in the polyadenylation signal that adds polyadenylic acid in the plant to the 3 ' end of RNA.The example in suitable 3 ' district is (1) 3 ' untranslated region of transcribing; contain for example polyadenylation signal of rouge alkali synthetase (NOS) gene of Agrobacterium tumor inducing (Ti) plasmid gene; (2) plant gene; as Semen sojae atricolor storage protein gene and ribulose-1,5-bisphosphate; 5-diphosphonic acid carboxylase (ssRUBISCO, SSU) small subunit of gene.

Plant Transformation/regeneration

When exploitation nucleic acid construct of the present invention, the various components of construct or its fragment will be inserted into cloning vehicle easily usually, for example in the plasmid that can duplicate in bacterial host such as E.coli.There are many carriers of having described in the literature, wherein many can buying from commerce.After each clone, the cloning vehicle with required insert can be separated, the single stepping of going forward side by side, and new fragment or nucleotide are inserted in for example Restriction Enzyme digestion, connects, disappearance, sudden change, excisions etc. are to adapt to the component of required sequence.In case construct is finished, it can be transferred in the suitable carriers then, is used for further operation according to the transform mode of host cell.

Recombinant DNA molecules of the present invention generally includes selectable labelling so that cell transformed can easily be identified and never choose in the cell transformed.Such example includes but not limited to neomycin phosphotransferase (nptII) gene (Potrykus etc., 1985), and it gives kalamycin resistance.The cell of expressing the nptII gene can use suitable antibiotic for example kanamycin or G418 selection.Other selectable markers commonly used comprise the bar gene, and it gives the bialaphos resistance; The epsp synthase gene (Hinchee etc., 1988) of sudden change, its conferring glyphosate resistance; Nitrilase gene, it gives Brominal (bromoxynil) resistance (Stalker etc., 1988); The acetolactate synthase gene (ALS) of sudden change, it gives imidazolone or sulphur urea resistance (european patent application 154,204,1985); With methotrexate resistance DHFR gene (Thillet etc., 1988).

The genetically modified plant of PBGS be can be used to express and Robinia pseudoacacia L., alfalfa, Fructus Foeniculi, Fructus Mali pumilae included but not limited to, Fructus Pruni, Carlina acaulis, rocket salad, Germinatus Phragmitis, American Avocado Tree, Fructus Musae, Fructus Hordei Vulgaris, bean, Radix Betae, blackberry, Pericarpium Citri tangerinae, Broccoli, Brassica oleracea L.var.gemmifera Zenk., Brassica oleracea L.var.capitata L., Brassica campestris L, cantaloupe, Radix Dauci Sativae, Maninot esculenta crantz., Brassica oleracea L. var. botrytis L., Herba Apii graveolentis, Fructus Pruni pseudocerasi, cilantro, Citrus, the little Citrus of Ke Laimenshi, coffee, corn, Cotton Gossypii, Fructus Cucumidis sativi, Pseudotsuga menziesii (Mirbel) Franco, Fructus Solani melongenae, Herba Cichorii, thatch dish (escarole), Eucalyptus, Fructus Foeniculi, Fructus Fici, calabash, Fructus Vitis viniferae, grapefruit, honeydew melon (honey dew), Pachyrrhyizus erosus, Fructus actinidiae chinensis, Caulis et Folium Lactucae Sativae, Folium Allii sativi, Fructus Citri Limoniae, Citrus aurantium Linn., torch pine, Fructus Mangifera Indicae, Fructus Melo, mushroom, nut, Herba bromi japonici, Brassica campestris L (oil seed rape), Flos abelmoschi manihot, Bulbus Allii Cepae, orange, ornamental plant, Fructus Caricae, parsley, Semen Pisi sativi, Fructus Persicae, Semen arachidis hypogaeae, pears, Fructus Capsici, Fructus Kaki, pinaster, Fructus Ananadis comosi, Herba Plantaginis, Fructus Pruni salicinae, Punica granatum L., Cortex Populi dividianae, Rhizoma Solani tuber osi, Fructus Cucurbitae moschatae, WENBO, pine (radiata pine), Chrysanthemum lettuce, Radix Raphani, Fructus Rubi corchorifolii Immaturus, Oryza sativa L., rye (Secale cereale L.), Sorghum vulgare Pers., south pine, Semen sojae atricolor, Herba Spinaciae, Fructus Cucurbitae moschatae (squash), Fructus Fragariae Ananssae, Radix Betae, Caulis Sacchari sinensis, Helianthi, Rhizoma Dioscoreae esculentae, sweetgum, Fructus Citri tangerinae (tangerine), tea, Nicotiana tabacum L., Fructus Lycopersici esculenti, black Semen Tritici aestivi, turf, rattan, Citrullus vulgaris, Semen Tritici aestivi, Rhizoma Dioscoreae, and Cucurbita pepo L. (zucchini).

The PBGS gene can be inserted in the Plant Genome by any suitable method.Suitable plant conversion carrier comprises the Ti-plasmids from Agrobacterium tumefaciems (Agrobacterium tumefaciens), and for example by (1983) such as Herrera-Estrella, Bevan (1984), Klee etc. (1985) and EPO disclose the carrier that discloses in 120,516.Except from the Ti-plasmids of Agrobacterium or the plant conversion carrier of root induction (Ri) plasmid, alternate method also can be used for DNA construct of the present invention is inserted into plant cell.Such method can comprise, for example, utilizes the chemicals of liposome, electroporation, the absorption of increase dissociative DNA, and the conversion of virus or pollen is sent and used to the dissociative DNA that utilizes microinjection to bombard.DNA can also be inserted into chloroplast gene group (Daniell etc., 1998).

The plasmid expression vector that is fit to use the microinjection bombardment to introduce the PBGS gene in monocotyledon comprises as follows: CTP; The light inducible promoter; The PBGS gene; The intron that the shearing site that is beneficial to gene expression is provided is Hsp70 intron (the open WO93/19189 of PCT) for example; With 3 ' polyadenylation sequence for example rouge alkali synthetase 3 ' sequence (NOS 3 '; Fraley etc., 1983).This expression cassette can be assembled on being suitable for producing the height copy replicon of a large amount of DNA in plant to be injected.

Being used to transform the useful especially plant conversion carrier based on Agrobacterium of dicotyledon is plasmid vector pMON530 (Rogers etc., 1987).Plasmid pMON530 is the derivant (Rogers etc., 1987) of the pMON505 for preparing by the fragment that shifts pMON316 2.3kb StuI-HindIII in pMON526.Plasmid pMON526 is the simple derivatives of pMON505, and wherein the SmaI site is by digesting with XmaI, and the Klenow polymerase is handled and removed with being connected.Plasmid pMON530 keeps all character of pMON505 and CaMV35S-NOS expression cassette, and comprises unique SmaI cleavage site now between promoter and polyadenylation signal.

Two carrier pMON505 are derivants of pMON200 (Rogers etc., 1987), and wherein Ti-plasmids homology zone LIH is replaced by the 3.8kb HindIII-SmaI fragment of miniature RK2 plasmid pTJS75 (Schmidhauser and Helinski, 1985).This fragment comprises RK2 replication origin oriV and shifts starting point oriT, is used for being attached in the Agrobacterium by triparental mating method (tri-parental matingprocedure) (Horsch and Klee, 1986).Plasmid pMON505 keeps all key characters of pMON200, comprise and be used to insert required segmental synthetic polylinker, the chimeric NOS/NPTII ' of plant cell kalamycin resistance/NOS gene, be used for strange unwrapping wire mycin/streptomycin resistance determinant in E.coli and Agrobacterium tumefaciems (A.tumefaciens) selection, be easy to the complete rouge alkali synthetase gene that the heritability among transformant and the offspring is assessed and be convenient in E.coli the pBR322 replication origin of a large amount of carriers of preparation.Plasmid pMON505 contain from pTiT37 nopaline-type T-DNA right-hand member single T-DNA border.The Southern hybridization analysis shows that plasmid pMON505 and its any DNA that carries are integrated in the Plant Genome, that is, whole plasmid is the T-DNA that is inserted in the Plant Genome.The end of the DNA that integrates is between right border sequence and rouge alkali synthetase gene, and the other end is between border sequence and pBR322 sequence.

Another useful especially Ti-plasmids boxlike carrier is pMON17227.This carrier is described among the open WO 92/04449 of PCT and contains the gene (called after CP4) of the enzyme of the conferring glyphosate resistance of encoding, and it is for comprising many plant excellent selectivity marker gene of Rhizoma Solani tuber osi and Fructus Lycopersici esculenti.This gene is fused to arabidopsis (Arabidopsis) EPSPS chloroplast transit peptides (CTP2) and passes through FMV promoter expression described herein.

When having obtained the cell that contains the PBGS gene (or plastid) of sufficient amount, this cell (or plastid) is reproduced in the whole plants.The selection of the method for regeneration step is not crucial, and for following host, suitable scheme is available: pulse family (Leguminosae) (alfalfa, Semen sojae atricolor, Herba Medicaginis etc.), Umbelliferae (Umbelliferae) (Radix Dauci Sativae, Herba Apii graveolentis, parsnip), Cruciferae (Cruciferae) (Caulis et Folium Brassicae capitatae, Radix Raphani, Brassica campestris L/Semen Brassicae campestris etc.), Cucurbitaceae (Cucurbitaceae) (Fructus Melo and Fructus Cucumidis sativi), Glumales (Gramineae) (Semen Tritici aestivi, Fructus Hordei Vulgaris, Oryza sativa L., corn etc.), Solanaceae (Solanaceae) (Rhizoma Solani tuber osi, Nicotiana tabacum L., Fructus Lycopersici esculenti, Fructus Piperis), the various crops that bloom, such as Helianthi, with tree glandiferous, such as Semen Armeniacae Amarum, Fructus anacardii, Semen Juglandis, and pecan.Referring to (1984) such as for example Ammirato; Shimamoto etc. (1989); Fromm (1990); Vasil etc. (1990); Vasil etc. (1992); Hayashimoto (1990); With (1990) such as Datta.

In one embodiment, the PBGS gene is from not comprising Mg ²⁺And comprise Zn ²⁺The species of PBGS enzyme.In preferred embodiments, these species are yeast or people.In another preferred embodiment, the PBGS gene of sudden change is used to produce transgenic plant.In another embodiment, the PBGS gene is introduced in the Plant Genome by homologous recombination.The wild type people PBGS genomic DNA and the full-length cDNA that can be used for generating transgenic plant are as follows:

People PBGS gene (SEQ ID NO:1):

cttacgcggtctgtgggagaccggagcgggagacagcggtgacaggagcagcggccgggagcccttagggaggcagacagagcctgcagccaatgccccaggagccctcggttccaaccaactgatgcccctgtgcccactggcccacgccatgcagccccagtccgttctgcacagcggctacttccacccactacttcgggcctggcagacagccaccaccaccctcaatgcctccaacctcatctaccccatctttgtcacggatgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtgaagcggctggaagagatgctgaggcccttggtggaagagggcctacgctgtgtcttgatctttggcgtccccagcagagttcccaaggacgagcggggttccgcagctgactccgaggagtccccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcctgtgatgtctgcctgtgtccctacacctcccatggtcactgcgggctcctgagtgaaaacggagcattccgggctgaggagagccgccagcggctggctgaggtggcattggcgtatgccaaggcaggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatcaaagaggccctgatggcacatggacttggcaacagggtatcggtgatgagctacagtgccaaatttgcttcctgtttctatggccctttccgggatgcagctaagtcaagcccagcttttggggaccgccgctgctaccagctgccccctggagcacgaggcctggctctccgagctgtggaccgggatgtacgggaaggagctgacatgctcatggtgaagccgggaatgccctacctggacatcgtgcgggaggtaaaggacaagcaccctgacctccctctcgccgtgtaccacgtctctggagagtttgccatgctgtggcatggagcccaggccggggcatttgatctcaaggctgccgtactggaggccatgactgccttccgcagagcaggtgctgacatcatcatcacctactacacaccgcagctgctgcagtggctgaaggaggaatgatggagacagtgccaggcccaagaactagaactttaaaacgttcccggggcctcagacaagtgaaaaccaaagtaaatgctgcttttagaactgtgccctcatgccctcttcctgctcacatgctagcggggcccagcagccctgggtggttttgccagcatgctaactcttgtaactcgcagctgcatcctatgagctctcccaagcttccccgcccctcccctgggtcagccgtgaggcccacctttgccaccctcagctctttcctctggtgtggcttcagcttgaaagcaacctggagtcgggggcacagcctttggggcctggctgggagagggtcttggagcattaggggaagaagagagcagtgggatcttggggcctgagaagccttggaacgcttctggcagcagagctgggtgtgggaatgaggcctagatcgatatccctgggttagagttgaaatttgccgcaattccactggaaggcatttcccacgaggccagaggttgccaggctgcctgaggtctcctattctactctgaaccataaacccagagaagaattactcattaaccagcataaatactgcctgaggatcaaaactcagaggcaaagagggagttcctgactgctagaggtgccaccaccacaaacactttttattcaggagatactttttgagaatctctgctctgttcctaggttcagtgctgggtcctgggaatacagcaggacagacctcagcttatctcttcatagaaattatacaaagagaattggggagacagctaagaagaaaacaaagaaataaagcagttacaaattgtgataagtgctttgaaggaaagaaggggtctgagacaacaacagggaaggggcctctcttgaaacagtagttgggaaggaggcagacatgcaccagtgatgtggtgacaggtgctctgaaggaggtcaccaggacctgacctctttgaaggatcagaaaatacttccctgaaggactgacatttgagcctagacctgaagggtgagccatcaagctaagacaattggggaagagcattccagggagagggaggagttgtgcaaaggccctggggctccttctagctggaggaatgcaaggctagcttgtctggagcactgagaggatggcctgaactgagtggagagagacagaccaggaccaaaccatgcagaggtcaagggccacattcaccttttcagagtgactcaatcaaatttgtagtttgtaaaagtattttaacagctctgcggcaaagtgcaaatgaaaagtcttgatggcatggactggagcggggacagtggggatggagaaaggggaatggattgtggatgtgtttagaaggtagattcgatgtgaaggatgaatctggcttgaccttctgggtggctgatgggccatttactgagatggggcagcctggaagaggaacagaagcagggtcggggtggagggagaatactaaacttagcttgagacattttgcaataaggaagctatatctagagtgcttatgtgactcacctaaggccactcaacaagtttgtggcagaactggattagaactgcacagaaaacagccaagctgggatttgaacccatgtagtccaactccaaggcctctgcccctaaccactgtgccataccacctcccaataatcaacagcaaaattataggtctaacaatgttttatagacacccctccatttatgtgatgggtttgcatcctgataaacccatcataagttgaaaatatgatcataagttgaaaatatgatcataagtcaaaaatgtatttaatatacctaacctaccaaacatcatagcttagcctagcctgccttaaacatgctcagaacacttacattagcctacagtgggcaaaactatccaacacaaaatctatattgtaataaagttgtaaagaattttgaataaaaattcaatatttgaaaaaaaaaaaaaaaaa

People PBGS cDNA (SEQ ID NO:2):

gcagccaaagccccaggagccctaggttccaaccaactgatgcccctgtgcccactggcccacgccatgcagccccagtccgttctgcacagcggctacttccacccactacttcgggcctggcagacagccaccaccaccctcaatgcctccaacctcatctaccccatctttgtcacggatgttcctgatgacatacagcctatcaccagcctcccaggagtggccaggtatggtgtgaagcggctggaagagatgctgaggcccttggtggaagagggcctacgctgtgtcttgatctttggcgtccccagcagagttcccaaggacgagcggggttccgcagctgactccgaggagtccccagctattgaggcaatccatctgttgaggaagaccttccccaacctcctggtggcctgtgatgtctgcctgtgtccctacacctcccatggtcactgcgggctcctgagtgaaaacggagcattccgggctgaggagagccgccagcggctggctgaggtggcattggcgtatgccaaggcaggatgtcaggtggtagccccgtcggacatgatggatggacgcgtggaagccatcaaagaggccctgatggcacatggacttggcaacagggtatcggtgatgagctacagtgccaaatttgcttcctgtttctatggccctttccgggatgcagctaagtcaagcccagcttttggggaccgccgctgctaccagctgccccctggagcacgaggcctggctctccgagctgtggaccgggatgtacgggaaggagctgacatgctcatggtgaagccgggaatgccctacctggacatcgtgcgggaggtaaaggacaagcaccctgacctccctctcgccgtgtaccacgtctctggagagtttgccatgctgtggcatggagcccaggccggggcatttgatctcaaggctgccgtactggaggccatgactgccttccgcagagcaggtgctgacatcatcatcacctactacacaccgcagctgctgcagtggctgaaggaggaatgatggaggacagtgccaggcccaagaactagaactttcaaacgttcccggggcctcagacaagtgacaaccaaagtaaatgctgcttttagaactgt

People PBGS aminoacid sequence (SEQ ID NO:3):

MQPQSVLHSGYFHPLLRAWQTATTTLNASNLIYPIFVTDVPDDIQPITSLPGVARYGVKRLEEMLRPLVEEGLRCVLIFGVPSRVPKDERGSAADSEESPAIEAIHLLRKTFPNLLVACDVCLCPYTSHGHCGLLSENGAFRAEESRQRLAEVALAYAKAGCQVVAPSDMMDGRVEAIKEALMAHGLGNRVSVMSYSAKFASCFYGPFRDAAKSSPAFGDRRCYQLPPGARGLALRAVDRDVREGADMLMVKPGMPYLDIVREVKDKHPDLPLAVYHVSGEFAMLWHGAQAGAFDLKAAVLEAMTAFRRAGADIIITYYTPQLLQWLKEE

Compositions of the present invention is suitable as the antimicrobial acivity composition in the personal care product, described personal care product is shampoo for example, the shower additive, hair care product, liquid and bar soap (based on synthetic surfactant and salt saturated and/or undersaturated fatty acid), emulsion and cream, eliminating smell agent, the solution of other aqueouss or alcohol, for example clean solution of skin, moistening cleaning cloth, oil or powder.Therefore the invention still further relates to carrier or adjuvant that optional beauty treatment that the United States Patent (USP) 6,689,372 of the personal care product that comprises the present composition and Holzl etc. describes can tolerate.Said composition will promptly suppress or prevent the amount use of microbial activity effectively to have anti-microbial effect.Can use other component, for example, chelating agen, coloring agent, perfumery oil; thickening or curing (denseness adjusting) agent, emollient, UV absorbent, Derma-Guard, antioxidant; the additive that improves engineering properties is the Al of dicarboxylic acids and/or fatty acid for example, Zn, Ca and Mg salt and optional antiseptic.In addition, the invention provides the method for skin, mucosa or hair antimicrobial therapy, comprise that skin, mucosa or the hair with the people of the described antimicrobial therapy of needs contacts with the present composition of antimicrobial effective dose.

Can be mixed with Water-In-Oil or oil-in-water emulsion according to personal care product of the present invention, alcohol or contain alcohol goods, the vesicle disperse system of ion or non-ionic both sexes fat, gel, solid rod or aerosol product.

As oil-in-water or water in oil emulsion, the adjuvant that beauty treatment can tolerate comprises for example oil phase of 5-50%, the water of 5-20% emulsifying agent and 30-90%.Oil phase can comprise any oil that is suitable for beautification product, one or more hydrocarbon ils for example, wax, natural oil, silicone oil, fatty acid ester or aliphatic alcohol.Preferred monohydric alcohol or polyhydric alcohol are ethanol, isopropyl alcohol, propylene glycol, hexanediol, glycerol and Sorbitol.

Can be included in according to beautification product of the present invention in U.S patent 6,689, the 372 described multiple beautification products of Holzl etc.Especially consider following goods, for example: the skin nursing goods, clean and cleaning article the detergent of no soap or washing ointment as the skin that adopts tablet or liquid soap form; The shower goods, for example liquid (shower foam, emulsion, shower goods) or solid shower goods are as bath cube and bath salt; The skin nursing goods, as the skin emulsion, multistage emulsion or skin oil; The beauty treatment personal care product is as adopting the facial color dress of day cream or Refined Mercurous chloride form, facial foundation (loose shape or compression), rouge (rouge) or vanishing cream (cream make-up), the eye-care goods are as the eye shadow goods, mascara, informer, eye cream or solid eye cream (eye-fix creams); The lip care article, as lip pomade, lip gloss, lip liner, manicure goods, nial polish for example, cleaner, fingernail hardening agent or epidermis remover; The secret hygienic articles is as secret washing liquid or secret spray; The foodcare goods, as foot bath liquid, foot-powder, sufficient breast or foot balsam, special eliminating smell agent and antiperspirant or remove the cocoon goods; Sunscreen, for example sun-proof milk, suntan lotion, sunscreen cream, and suntan lotion, sunscreen or tropicals, tanned preceding (pre-tanning) goods or solarization back care article; The skin tanning goods are as self-service solarization black frost; Remove the pigment goods, as the goods or the skin glow goods of bleaching skin; Anthelmintic, as anthelmintic oil, insect repellant solution, anthelmintic spraying or anthelmintic rod; Eliminating smell agent, for example taste removal spraying, the spraying of pumping action, taste removal gel, taste removal rod or taste removal ball; Antiperspirant, as the hidroschesis rod, hidroschesis frost or hidroschesis pearl; The goods of cleaning and nursing skin speckle are as no soap detergent (solid or liquid), frosted goods or frosted facial film; The depilation goods of chemical species, as depilating powder, liquid depilation goods, the depilation goods of frost or cream form, the depilation goods of gel form or aerosol foam; The goods that shave, as shaving soap, foam shaving cream, still shaving cream, goods before foam and the gel, the dried palpus that shaves, aftershave lotion or must back liquid; Flavor article is as spice (GULONG water (eau de Cologne) washes water (eau de toilette), and light perfume washes with perfume (parfum de toilette) perfume), the fragrant atmosphere of essential oil or mastic; Dental care, artificial tooth nursing and mouth care goods, as toothpaste, gel dentifrice, dentifrice concentrates collutory, anti-plaque collutory, denture cleanser or artificial tooth sticking agent; Beauty treatment hair treatment goods, as the hair washing goods of shampoo or hair conditioner form, the hair nursing goods are as the pretreatment goods, the hair moisturizing agent, moulding frost, moulding gel, brilliantine, the hair purificant, pack processing (treatment packs), reinforced hair treatment, hair construction goods, as the hair wave goods (burn, warm the scalding that produce lasting wave, cold wave), the straight goods of hair, liquid hair consolidated article, foam, hair spray, bleaching goods; As hydrogenperoxide steam generator, luminous shampoo, bleaching frost, bleaching powder, bleaching cream or oil, temporary transient, semi-durable or persistent hair coloring agent contains the goods of autoxidation dyestuff or natural hair coloring agent, for example Flos Impatientis (henna) or Flos Chrysanthemi.

According to oral composition of the present invention can be, for example, gel, cream, frost or water-based product (collutory) form.

Can also comprise the chemical compound that discharges the fluorion of effectively resisting dental caries formation according to oral composition of the present invention, inorganic fluoride salt for example, as sodium fluoride, potassium fluoride, ammonium fluoride or calcium fluoride, or organic villiaumite, as amine fluoride, it is known with trade name Olafluor.

Compositions of the present invention also is suitable for handling textile fibre materials.Such material is as silk, Pilus Caprae seu Ovis, undyed and the painted or stamp fibrous material of polyamide or polyurethane and especially various cellulosic fibre material.Such fibrous material is, for example, and native cellulose fibre, for example cotton, Caulis et Folium Lini, Corchorus olitorius L. and Fructus Cannabis, and cellulose and regenerated cellulose.Preferred suitable textile fiber material is made by cotton.Compositions of the present invention can also be used for washing and cleaning article, as is used for liquid or solid detergent or softening agent.

Compositions of the present invention also is fit to give plastics with anti-microbial properties, described plastics such as polyethylene, polypropylene, polyurethane, polyester, polyamide, Merlon, latex etc.Therefore the field of using is, for example, and floor covering, plastic coating, plastic containers and packaging material, kitchen and bathroom apparatus (as, brush, shower curtain, sponge, slipmat), latex filter material (air and water filter), the plastic article that is used for field of medicaments, as dressing, syringe, conduit etc., so-called " medical apparatus and instruments ", glove and mat.

Paper for example is used for the paper of health purpose, can also use compositions of the present invention to make it have anti-microbial properties.

According to the present invention, make non-woven fabrics, as diaper, sanitary towel, it also is possible that close-fitting lining (panty liners) and the cloth that is used for health and household have anti-microbial properties.

Said composition can also be particularly useful for household and all-round cleaning agent with the cleaning and disinfection crust.

Except anticorrosion to cosmetics and family product, technical products is the printing thickening agent of paper treatment fluid, starch or cellulose derivative for example, and face coat and coating also can be by anticorrosion and have an anti-microbial properties.

Compositions of the present invention also is suitable for handling and making leather have anti-microbial properties to the antimicrobial treatment of timber with to the antimicrobial thing of leather.

Chemical compound of the present invention also is suitable for protecting cosmetic product and family product to avoid the microorganism damage.

In addition, compositions of the present invention can also be used as oral composition, White for example, the U.S. patent 6,740,311 of Jr. etc. that describe with the bonded dentifrice composition of oral cavity acceptable carrier.The nonrestrictive example of this oral composition is the toothpaste that is applicable to humans and animals, dentifrice, preventative toothpaste, lozenge, chewing gum etc.

In addition, compositions of the present invention can be used to prepare antimicrobial surface.The method for preparing antimicrobial surface is provided in addition, this method comprises: (1) provides compositions of the present invention, wherein thereby said composition effectively suppresses or prevents to form the activity form of polymer porphobilinogen synzyme and suppress or prevent antibacterial, archeobacteria and/or Eukaryotic growth or growth, and it is at least a in medicine, toothpaste, soap, disinfectant, antibiont film composition and the herbicide that the activity form of supposing polymer porphobilinogen synzyme comprises the magnesium binding site of allosteric and said composition; (2) provide the surface to form substrate; (3) thus compositions and surface are formed matrix group merges preparation antibacterium surface.In a variant of this method, the antibacterium surface is adapted to prevent or suppresses biomembranous formation.

Term used herein " surface form substrate " comprises biodegradable and nondegradable polymer, Silicon stone, pottery and makes up, and is used for compositions with substrate mixing, layering or be connected.Said composition can also place the top or the lower surface of substrate.

In the present invention, the method of handling plant growing or growth is provided, comprise plant used compositions of the present invention as herbicide, wherein this plant be Herbicid resistant and be adapted to being genetically modified to embrace the polymer porphobilinogen synzyme that dimeric polymer form exists substantially.In a variant of this method, polymer porphobilinogen synzyme does not contain the magnesium binding site of allosteric.

The present invention will explain in more detail by the following embodiment of being, but be to be understood that and the invention is not restricted to this.

Embodiment

As an example porphobilinogen synzyme or MORPHEINS:

The following examples have disclosed PBGS can be with the architecture basics that alternate quarternary structure state exists and the mutual conversion of these stastus formats forms these PBGS allosteric regulation and control in some species.The known quarternary structure state of PBGS is eight aggressiveness, constitutes by embracing dimer.Those of QSE among also known PBGS, especially Fig. 6 exist with the balance that contrasts the shown quarternary structure form of active protein concentration dependency.Contrasting oligomer that active protein concentration dependency shows maximum activity can dissociate or heavily be combined into the lower form of littler activity.Think the form that littler activity is lower, also be to embrace dimeric plural form before.The PBGS that is in QNW is not easy between the quarternary structure isoform equilibrated fact makes observation and sign to the dimeric stable oligomer that separates become possibility.Therefore, the F12L of people PBGS sudden change allows us to study the stable form of six aggressiveness and has set up just that the character of its six aggressiveness (rather than specific F12L sudden change) has determined the functional character of F12L with respect to the strong discrepancy of wild type people PBGS.F12L is the rare allele (3-5) of the natural generation of people PBGS.What describe below is research (in E.coli the wild type of heterogenous expression and F12L0 and by routine techniques purification) to people PBGS.

Protein expression

Parent people PBGS is the N59/C162A (6) that fully characterizes.N59 corresponding in coding PBGS proteic two codominant alleles more soluble that.C162A eliminates the optimum sudden change that slowly forms the probability of unusual disulfide bond.Be called Wt below the artificial gene of N59/C162A.Being used for the sense strand primer that Wt QuikChange sports the F12L variant is GGCTACCTCCACCCACTGCTTCGGGCC.Several constructs prepare for coexpression Wt and F12L in E.coli.The order of gene all is different with the number of promoter, but these variations do not influence the result.The Wt that contains under the control of promoter and the construct of F12L have been described.The plasmid DNA (pET3aWt) that contains Wt digests to cut out Wt with BamHI and NdeI.The pET17b carrier DNA digests and linearisation by using BamHI and NdeI, and is connected with Wt, thereby the ATG start codon of Wt is positioned at 6 the base pair places, ribosome binding site downstream by vector encoded.The plasmid that obtains is transformed among the E.coli XL1blue.Preparation plasmid DNA (pET17bWt) is also used SpeI and Bpu1102I linearisation.The plasmid DNA (pET3aF12L) that contains gene F12L contains the fragment of ribosome binding site and the gene of F12L with generation with XbaI and Bpu1102I digestion.F12L gene and linearizing pET17Wt carrier are connected 35 base pairs in termination codon downstream that make the ribosome binding site of F12L gene be positioned at Wt, and terminator is positioned at F12L gene termination codon downstream 52 base pairs.Plasmid pET17bWtF12L is transformed into E.coli XL1blue, and the preparation plasmid DNA also transforms E.coli BLR (DE3) with expressing protein, (6) as previously mentioned.

Protein purification

The big portion of method for purifying proteins (cell breakage, ammonium sulfate precipitation, phenyl-Sepharose hydrophobic chromatography, ion-exchange chromatography and Sephacryl S-300 gel permeation chromatography) follows the method for aforementioned (6), replaces being used for the DEAE agarose column of anion exchange step except using 70ml Q-Sepharose post.Q-Sepharose at room temperature uses the 30mM potassium phosphate, and pH 7.0, the 10mM 2 mercapto ethanol, and 10 μ M Zn (II), and adopt KCl gradient as shown in Figure 3A to carry out.Gradient is that 3ml min-1 is by control of Rainin HPLC system and collection 10ml fraction with the flow velocity.

The dynamic characteristic of PBGS variant is used to show that wt and f12L have different functional characters:

At 0.1M bis-tris propane, the 10mM 2 mercapto ethanol carries out all kinetic determinations among the 10 μ M Zn.For pH variation diagram (rate profile), adding the pH reflection analysis pH that reports behind the 10mMALA-HCl.Km and Vmax are measured, and the concentration of ALA is 10 μ M, 30 μ M, and 100 μ M, 300 μ M, 1mM, 3mM, and 10mM, and respectively repeat twice.The variation of ALA-HCl concentration does not cause the variation of final pH because add constant volume in the analysis of mixtures before, 0.1M ALA-HCl liquid storage is diluted among the 0.1M HCl.All analyses use EhrlichShi reagent to measure the porphobilinogen that forms 37 ℃ one period regular time.

Analytical ultracentrifugation:

Protein sample only last sample to super centrifugal before to the 30mM potassium phosphate, pH 7.5,0.1mMDTT and 10 μ M ZnCl ₂Dialysis.Last sample concentration is respectively 10.6 μ M and 12.8 μ M to wild type and F12L mutant enzyme.Be equipped with the Beckman OptimaXL-A analytical ultracentrifuge of An60Ti rotor and use 6 passages 4 ℃ of uses, the 12-mm path, the Epon central part of filling active carbon carries out whole sedimentation equilibrium experiments with quartz window.Three spinner velocities (8,000,11,000 and 14,000rpm) collect data and use the 0.001cm scanning step to show average 20 scannings.Use Sednterp program (32) gauged little partially specific volume of accounting temperature (partialspecific volume) and solution density; Solution concentration is 1.00191gm/mL, and little partially specific volume of wild type and mutain is respectively 0.7394 and 0.7397mL/gm.Use is from University of Connecticut (University of Connecticut) (Storrs, the HID program analysis data of analytical ultracentrifugation facility CT).Having got rid of single species from match (fits) obviously is nonrandom as the digital simulation analysis of residue.

The mensuration of F12 crystal structure:

F12L is to 50mM bis-tris propane, and 10mM β ME and 10 μ μ M ZnCl2 dialyse.Use heavy (sitting drop) method formation crystal equal-volume F12L (4.0mg ml-1) that drips to mix with precipitant (0.4M Ammonium biphosphate).To mole ALA such as protein protomer concentrated solution interpolations, in 3-5 days, form crystal.On the MAR345 imaging plate detector that is connected with the RU-200 rotation anode generator that has assembled the OSMIC optical element and worked under 50kV and the 100ma, collect diffraction data with 100K.Before freezing by crystal is transferred in each solution, contain 12%, 17%, 23% and the liquid storage of 30% glycerol in 3 minutes and this crystal of cryoprotection.Some data show height of collecting are unordered and lack any part between the active region.Because above-mentioned factor, the crystal of F12L is soaked in 2mM ALA (it is added to the end in two cryoprotection solution) and 0.2mM ZnCl2 (it also is added in latter two solution except ALA).Final data set is made up of 525 frames corresponding to 0.5 ° of vibration, 3.5 minutes open-assembly times of every frame.Crystal belongs to hexagonal system, spatial group P63, unit cell parameter a=b=89.6 , c=153.2 .Two kinds of molecules are arranged in asymmetric unit.Diffraction data program package HKL2000, Rmerge (I)=5.0% pair 33,615 are reflected in 45-2.2 resolving range and simplify.

Carry out molecular replacement with the molecule A of people PBGS structure (pdb encode 1E51) as initial model by the AmoRe program package and determine structure.Improve with the CNS program.Final mask comprises F12L-molecule A (residue 11-82,97-124,140-169,172-212,222-330) and B (residue 3-82,97-122,140-169,172-212, dimer 226-328), be combined in a molecule of the catalytic reaction intermediate product of molecule A avtive spot, 241 hydrones and seem to have two zinc atoms that low orbit occupies.For 2.2 differentiated delta data, the R-factor of crystal imaging was 19.9%, and R (free) is 28.6%, and the RMS of bond distance and bond angle departs from and is respectively 0.18 and 2.0 °.All residues belong to the configuration zone that allows on the Ramachandran figure.

The character of people PBGS variant F12L

People PBGS variant F12L is significantly different with wild-type protein.The feature of the F12L of purification confirms that its catalytic activity is very low under the highest condition of wild type people PBGS activity.Yet, F12L quite high activity (Figure 1A) when showing the pH variation diagram that obviously changes and being presented at alkaline pH value.Proteic at wild type just when the K that measures F12L and wild type people PBGS of pH 7 and F12L just when pH 9 _mAnd V _MaxValue; The results are shown in table 1 (hereinafter).F12L shows normal Michaelis-Menten kinetics and unusual high K _mValue is far above the physiological concentration of substrate 5-aminolevulinic acid (ALA).Yet, the V of F12L when pH 9 _MaxBe significantly higher than the V of wild-type protein _MaxIn optimum pH with exist under the condition of the suitableeest configuration of metal ion, the wild type PBGS of all feature known species its K that is in the news _mValue 100 μ M (6,8-10) in the scope, be in the value of 7 times finding wild types of pH people PBGS as this.Wild type people PBGS shows the Michaelis-Menten kinetics that does not have display standard in the kinetics of pH 9, and its basis is also not obvious at the beginning.According to rough inspection, wild-type protein seems to show with Hill coefficient 0.35 magnitude and shows extreme negative cooperativity.In fact, are curve equation in pairs to the best fit of these data, it was considered to come from the catalytic action of quarternary structure isoform (morpheins, eight aggressiveness and six aggressiveness) mixture afterwards, and wherein two kinds of forms have different Km values.This phenomenon is more detailed hereinafter to be described.

The further evidence of significant difference is from anion-exchange chromatography (Figure 1B) and native gel electrophoresis (Fig. 1 C) mobility's variation between F12L variant and the wild-type protein, and the both hints the difference of oligomer structure.Separation on anion-exchange column reflects different surface charges usually, its can not be since neutral leucine by the replacement of neutral phenylalanine.The electrophoretic separation of two kinds with identical lotus/matter ratio shows or varies in size or the shape difference.Generally speaking, these differences show that F12L exists with different oligomerization states with wild type people PBGS.

The super centrifugal sedimentation equilibrium analysis of carrying out of the albumen operational analysis of wild type and sudden change, the molecular weight of wild-type protein and F12L is found and is respectively 244,000 ± 8, and 900 and 197,900 ± 6,500 dalton.The former expects eight aggressiveness and six aggressiveness midway, and the latter is expection six aggressiveness and midway tetrameric.In the model analysis of data, wild-type protein is respectively with the model of 7.6%, 51% and 42% best fit to dimer, six aggressiveness and three states of eight aggressiveness, and F12L is with 70% to 30% the ratio best fit model to two states of the tetramer and six aggressiveness, and do not have eight aggressiveness.Therefore, the inventor has measured the crystal structure of people PBGS variant F12L.

The significant difference of the crystal structure displaying monomer structure of people PBGS and F12L variant, first example that this determines new quarternary structure isoform and has disclosed MORPHEINS:

The crystal structure of 17 PBGS from fungus, metazoa and antibacterial (11-20) that measured in the past shows total homotype eight aggressiveness structures, and wherein four dimers are by be correlated with around 90 ° of rotations of the axis of centres (Fig. 2 A).PBGS is the member of TIM α/β tubbiness albumen (21) aldolase superfamily.Catalytic core is positioned at bucket fully in each subunit, and the terminal arm of 20+ amino acid N relates to widely, and subunit interacts.The sequence of catalytic core is conservative on the phylogenetics, but the N-terminal arm is not conservative.PBGS dimer seen in eight aggressiveness (Fig. 2 A, top) comprises that the bucket of high conservative embraces the bucket of sister's subunit to the N-terminal arm of a bucket contact and a subunit.Therefore, this is called as and embraces dimer (hugging dimer) (2).The side chain of amino acid/11 2 does not participate in embracing interaction.Tetrameric set, its by adding around the axis of centres (Fig. 2 A, centre) half-twist second embraces dimer, and adds interact with each other between the arm of a subunit and the adjacent dimeric α/β drum head portion.The side chain of amino acid/11 2 participates in this subunit and interacts.Add two again and produce eight aggressiveness (Fig. 2 A, bottom) around the dimer of axis of centres half-twist separately.This eight aggressiveness towards reader's half-twist, produces the expression of windmill with respect to dimer and tetrameric view.Before the crystal structure of determining F12L, suppose that all PBGS albumen has identical homotype eight aggressiveness structures (2).Yet for the PBGS from green plants and some antibacterials, the active eight maximum aggressiveness of kinetics evidence hint can be dissociated into little, active lower construction unit (9,22).This kinetics evidence is the active protein concentration dependency of contrast shown in Semen Pisi sativi PBGS among the figure X.

Noticeablely be that the new crystal structure of measuring (PDB numbers 1PV8) of F12L people PBGS allele has shown and comprises the quarternary structure (Fig. 2 B) of N-terminal arm with respect to the remarkable rearrangement of α/β bucket.In this case, dimer keep above-mentioned bucket to the bucket contact but the N-terminal arm is (Fig. 2 B, top) separately rather than that embrace.Tetrameric set keeps the interact with each other of the arm of an above-mentioned subunit and adjacent dimeric α/β drum head portion.Yet because arm is outstanding, this connection has been stipulated around 120 ° of rotations of the axis of centres.Therefore, three dimers that separate are arranged in the oligomer structure, rotate 120 ° around the axis of centres separately and form six aggressiveness (Fig. 2 B, bottom are represented to observe with windmill).Are fabulous examples from observed wild type people PBGS eight aggressiveness to the unprecedented structure conversion of six aggressiveness of observed F12L, illustrated how little sudden change has deep effect to proteic 26S Proteasome Structure and Function, and shown that how approaching these two kinds of quarternary structure forms are on energy.Must be undertaken by the mutual conversion of embracing dimer and separate between the dimer in conjunction with knowing by observing these in any balance between eight aggressiveness and six aggressiveness.This mutual transformation process is shown in Fig. 5 A.

The new construction of F12L (2.2 resolution) comprises significant disordered regions, and it makes and can't carry out with respect to the structure comparison of the avtive spot of aforementioned wild type people PBGS structure (PDB numbers 1E51,2.83 resolution).Amino acid/11 2 directly with arbitrary structure in the avtive spot residue interact.In addition, for all observed those aminoacid in two structures, great majority are can be eclipsed.Therefore, for the basis of further surveying the unusual kinetic property of F12L (as, Fig. 1, table 1), the inventor's coexpression F12L and wild type people PBGS.

It is the basis of kinetics difference that the coexpression of wild type people PBGS and F12L discloses quarternary structure:

Preparation coexpression system system is to produce 1: 1 wild type people PBGS and F12L variant from identical RNA courier.The purification of the albumen WT+F12L of coexpression is found in and produces two different PBGS protein peaks (Fig. 3 A) in the anion-exchange chromatography.At first the peak of eluting (pond I) is suitable with F12L swimming on native gel, and second peak (pond II) and wild type people PBGS swimming suitable (Fig. 3 B).Pond I is presented at pH 9 increased activity and pond II is presented at pH 7 increased activity (Fig. 3 C).Two ponds carry out mass spectral analysis respectively and are found terminal 2010.2 dalton of the N-that contains significant quantity separately containing the peptide of Phe and the peptide that 1976.2 dalton contain Leu behind trypsinization, confirmed that two ponds contain the heteromers kind.The percentage ratio of each chain comes quantitatively by N-terminal order-checking in the heteromers pond, shows that pond I contains 48.5%Phe and 51.5%Leu and pond II contains 71.1%Phe and 28.3%Leu.It is the quarternary structure what has dominated the heteromers kind that these ratios do not have obviously to show.Pond I and II are by being further purified at Sephacryl S300 gel filtration, and this has reduced the cross-contamination of heteromers.The pond I of S300 purification and the pH variation diagram of II respectively with F12L and wild type people PBGS significantly similar (Fig. 3 C).According to chromatography, mass spectrum and quantitative N-terminal sequencing data, we sum up that pond I comprises assorted six aggressiveness and pond II comprises assorted eight aggressiveness.It is more by quarternary structure control rather than be in the 12nd aminoacid and form and control that the pH variation diagram is found.

Measure the kinetic parameter Km and the Vmax (table 2) in the pond of S300 purification at pH 7 and pH 9.Dynamics data is not followed simple Michaelis-Menten relation (hyperbolic fit), but can ascribe the catalysis (curve fitting in pairs) (23) by two kinds of multi-form enzymes with different Km and Vmax value to.Fig. 3 D shows the activity as the concentration of substrate function; The even model of fit of dynamics data, wherein six aggressiveness of enzyme and eight aggressiveness forms show high and low Km value respectively.This curve fitting in pairs (dark line) is much better than single hyperbolic fit (light line).All kinetics values are all accurately measured, exception be to detect pond I at pH 9 to have trace eight aggressiveness (referring to table 2).Also to be provided than the better match of eight aggressiveness-six aggressiveness model, this solution is also included within table 2 to the data of wild type people PBGS during pH 9.The equilibrated factor of control people's PBGS heteromers (heteromer) is still waiting to illustrate under analysis condition.

Table 2 wild type people PBGS, the kinetic parameter in F12L and heteromers Wt/F12L pond

		F12L	Wild type	WtF12L pond I	WtF12L pond II
						K m1	pH 7		0.25±0.01	0.21±0.01	0.13±0.01
V max1	pH 7		55.5±0.2	2.37±0.07	20.20±0.99
						K m2	pH 7	17.7±1.1		7.71±0.72	4.85±2.04
V max2	pH 7	1.14±0.05		4.78±0.10	10.92±0.85
						K m1	pH 9		0.35±0.09	0.10±0.13	0.024±0.003
V max1	pH 9		8.16±0.13	0.32±0.23	3.19±0.15
						K m2	pH 9	4.6±0.1	4.46±0.80	3.74±0.29	2.35±0.21
V max2	pH 9	18.2±0.2	6.67±0.36	12.16±0.18	8.70±0.17

Pond I and pond II are as shown in Figure 3A, further active two ponds of the PBGS of purification on Sephacryl S-300 post behind the Q-Sepharose post eluting.K _M1And K _M2(all being mM) is interpreted as the K of eight aggressiveness and six aggressiveness respectively _mThe V of report _MaxValue (the μ molesh of unit ^-1Mg ^-1) be reflected in that the quarternary structure kind has molar fraction to be determined under the analysis condition.The K of match _mValue is independently to the distribution of quarternary structure kind.

Wild type people PBGS, the F12L variant, and the data that present of the different aggressiveness of WT+F12L have formally been set up kinetics difference between wild-type protein and the F12L variant mainly due to the difference of quarternary structure.Select the further work of people's PBGS mutants (R240A, T23P, and T23P/F12L) to confirm that the kinetics of six aggressiveness is similar to the kinetics of F12L and the kinetics of eight aggressiveness is similar to the kinetics of wild-type protein to other.

Based on the understanding of eight aggressiveness, can conceive in hypothesis to the significant difference under the optimum pH of these two kinds of form PBGS to the people PBGS structure of six aggressiveness.The chemical property of the catalytic reaction of PBGS need form at least two Schiff alkali intermediate products (2,12,16,17,20).The amino that the formation of the carbinolamine of these Schiff alkali (carbinolamine) precursor requires to participate in is uncharged, and perhaps local pH is higher than amino pK _aA remarkable architectural difference between six aggressiveness and eight aggressiveness PBGS is the order degree of finding in the aminoacid that comprises avtive spot lid (lid).Six aggressiveness PBGS F12L crystal structures lack the compactness of the most of residues that constitute the avtive spot lid, thereby point out six aggressiveness structures to make the lid configuration of closing go to stablize.From body separated from solvent avtive spot, the catalytic reaction of PBGS can not be carried out under the situation that lacks the lid of closing, and is higher than the pK of the amino that participates in formation Schiff alkali up to outside pH _aTherefore, six aggressiveness structures be considered to have only when outside pH enough alkalescence so that just show activity when forming Schiff alkali.High Km also can ascribe to the avtive spot lid go stablize because the crystal structure of PBGS eight aggressiveness is presented at the stability interaction between the substrate molecule of the residue of lid and decision Km value.Present result provides the new approach of understanding regulation and control PBGS function.As mentioned below, to rethinking the active allosteric adjusting of inhuman kind PBGS considerable meaning is arranged from identifying the knowledge that PBGS six aggressiveness obtain.

The allosteric of PBGS is regulated can ascribe the balance of eight aggressiveness to six aggressiveness to:

The basis of relatively having disclosed the adjusting of PBGS allosteric of PBGS eight aggressiveness and six aggressiveness.Although in fact the obvious component of PBGS avtive spot is included in the monomer, most of PBGS albumen contain the binding site of the magnesium of allosteric, and this site is positioned at embraces dimeric arm to bucket (14,24) at the interface.All as seen, Fig. 4 A latter shows in the crystal structure of Pseudomonas aeruginosa (14) and E.coli PBGS (16) in the position of the magnesium of allosteric.What Fig. 4 A showed magnesium with the allosteric shown in the black ball embraces dimer (light color band, dark chain), and one of them shows with big black and white (white-on-black) arrow.The structure of yeast and people PBGS shows that arginic guanidine radicals is positioned at the magnesium place of allosteric, (2) as previously mentioned.This is the Arg240 of people PBGS.If supposing all PBGS can exist with six aggressiveness under appropriate condition, the position of the magnesium of allosteric is relevant with the conversion of six aggressiveness-eight aggressiveness so, is not present in six aggressiveness (constituting by separating dimer) because this melts combine site is present in eight aggressiveness (constituting by embracing dimer).Three subunits in Fig. 4 B demonstration PBGS eight aggressiveness are to the interface between the subunit.The black and white arrow shows the interface of bucket to bucket, and it is that eight aggressiveness and six aggressiveness PBGS set are common.The arrow of band stain (dot on black) shows the interaction of arm to drum head, and it also is that eight aggressiveness and six aggressiveness PBGS set are common.The black and white arrow, it is similar to the magnesium binding site of allosteric, shows that the arm be present in eight aggressiveness (embracing dimer) and be not present in six aggressiveness (separation dimer) interacts to bucket.With the magnesium of allosteric mediate six aggressiveness-eight aggressiveness equilibrated identical of views be the influence of magnesium to E.coli PBGS kinetic parameter.In this case, the magnesium of interpolation allosteric causes K _mValue is reduced to～200Mm (8) from～2mM, and this obviously points out the K of the people PBGS of six aggressiveness and eight aggressiveness forms _mDifference (table 1) between the value.Also it should be noted that our observation before, the pure E.coli PBGS that is homogeneous shows a plurality of bands in native gel electrophoresis, the swimming of these bands is consistent with eight aggressiveness, six aggressiveness and dimeric molecular size, helps forming maximum (eight aggressiveness) form (8) and add magnesium.It should be noted that also recent findings people PBGS variant R240A purification～80% is that six aggressiveness and 20% are eight aggressiveness, then an oligomer is unsettled and along with time rearrangement is six aggressiveness.

The observation of the specific activity that protein concentration is relied on is the most direct equilibrated diagnostic tool that has MORPHEINS:

The mutual conversion of PBGS between six aggressiveness and eight aggressiveness is proposed the mechanism of the specific activity that relies on as the protein concentration of being responsible for some species PBGS.Up to now, we have characterized four kinds of different PBGS of the magnesium that contains allosteric.Described enzyme is from species escherichia coli (γ-proteobacter), the living slowly root nodule bacteria of Japan (α-proteobacter), Pseudomonas aeruginosa (γ-proteobacter), and Semen Pisi sativi (Pisum sativum) (green plants).The back is different from the specific activity character (9,22,25) that people PBGS part is that they do not use the also total uncommon protein concentration in the catalytic zinc of avtive spot (24) and they to rely on for three kinds.Back one character shows that the oligomer of maximum activity can be dissociated into than low activity or do not have less form alive.The mathematical model of publishing has considered that eight aggressiveness of maximum activity are dissociated into than low activity or do not have the tetramer alive and/or dimer (9,22).

The six aggressiveness structures of people PBGS variant F12L make us advise that the protein concentration dependency of plant and some antibacterial PBGS is because than the balance between low activity six aggressiveness forms and the greater activity eight aggressiveness forms, shown in Fig. 5 A.Equilibrated existence like this obtains the support (data are not delivered) of Semen Pisi sativi PBGS sedimentation equilibrium research.Because magnesium is absolutely necessary to the difference of embracing between dimer and the alternate separation dimer, this ion is proposed and helps embracing dimeric formation, thereby and helps forming eight aggressiveness.Fig. 5 B example is removed magnesium ion from Semen Pisi sativi PBGS and is unfavorable for maximum form and helps less form, and wherein the swimming of two kinds of forms and eight aggressiveness and six aggressiveness is consistent.In this model, six aggressiveness are the potential storage form of PBGS albumen, because its activity under physiological pH is lower, and are characterised in that K _mValue, this value is far above the physiological concentration of ALA.In contrast, eight aggressiveness are active and its K in physiological pH _mValue is in the proper A LA concentration range in active tetrapyrrole biosynthetic process.

In a word, viewpoints of supporting of these researchs are PBGS work in the biosynthetic complicated regulation and control of chlorophyll (26-28).We notice that record shows and turn in the green process plant that magnesium density sharply increases (29) in the generation chloroplast.The six aggressiveness storage form that can imagine non-activity allow the fast activating of PBGS as following the part that turns the cascade reaction that biochemistry changes in the green process.What is interesting is that several gel filtration research summary oligomer of noticing plant and algae PBGS quarternary structure are six aggressiveness (30 and wherein quote document).Support to exist document in previously to report (31), to find from chlorella vulgaris (Chlorella regularis) PBGS by the separable PBGS level Four form that can change mutually of anion-exchange chromatography.

Six aggressiveness people PBGS show that allosteric is regulated the new structure example of protein function and are proteic first examples that exists as MORPHEINS:

Sign to people PBGS variant F12L discloses the huge change that point mutation causes the PBGS 26S Proteasome Structure and Function.The precedent that the single amino acids that the albumen behavior significantly changed during this sudden change can be used as and causes evolving changes.The F12L sudden change makes PBGS eight aggressiveness go stable and causes forming six aggressiveness.Structure conversion between eight aggressiveness and six aggressiveness must be undertaken by the balance beyond example that contains different dimeric structures.The magnesium that appears at the allosteric of most of PBGS has binding site in eight aggressiveness, and does not have in six aggressiveness.The native gel data show that the magnesium of removing allosteric helps forming six aggressiveness, rather than eight aggressiveness.The conversion of eight aggressiveness-six aggressiveness has defined the new mechanism that metal ion dependency allosteric is regulated protein function.

The invention describes albumen that non-activity morphein by stablizing PBGS and/or other can regulate and control by the mutual conversion of morphein and the Profilin function.Optionally be attached to and the molecule of the six aggressiveness forms of stable PBGS for deciphering, the inventor takes following method.Only those PBGS at QSE are considered as target at present, have activity because these PBGS have shown as eight aggressiveness, but they show the specific activity phenomenon that protein concentration relies on.Target molecule is a kind of molecule that is selectively bound to " axillary fossa " of six aggressiveness shown in the ball among Figure 10.The inventor adopts " computer (in silico) " method retrieval molecular library to seek the molecule that is attached to the biological PBGS six aggressiveness forms of target.

Six aggressiveness PBGS set up the homology model for target

The crystal structure of unique existence of setting up target six aggressiveness PBGS models of inventor institute foundation is the clinical variant F12L of people PBGS, and PDB numbering 1PV8 (Breinig etc. (2003) Nat.Struct.Biol 10,757-763).Unfortunately, the crystal structure of F12L shows significant randomness, has limited it as unique basis of setting up the homology model.Yet, the relatively demonstration of people PBGS eight aggressiveness and six aggressiveness structures (PDB numbering 1E51 and 1PV8) is comprised TIM sample α, β barrel structure territory～300 aminoacid are almost consistent.For people PBGS, the difference of eight aggressiveness and six aggressiveness is the structure of 24 amino terminal amino acids and more unordered zones of different (referring to Breinig etc.) in six aggressiveness.Therefore, can use higher-quality PBGS eight aggressiveness crystal structures to set up the α of target PBGS, the homology model in β barrel structure territory.The structure of selecting is PDB numbering 1GZG (Frere, F., Schubert, W.D., Stauffer, F., Frankenberg, N., Neier, R., Jahn, D., and Heinz, D.W. (2002) J Mol Biol 320,237-247) list of references (20), it is the high-resolution crystal structure of Pseudomonas aeruginosa PBGS high-sequential, itself is the target of the inhibitor of " catching " PBGS six aggressiveness.Use the Swiss-PDB reader (www.expasy.ch/spdbv/mainpage.html) of different capabilities and the six aggressiveness forms that other programs are set up Pseudomonas aeruginosa PBGS.For setting up Pseudomonas aeruginosa PBGS six aggressiveness, from the dimeric structured file of 1GZG, remove the terminal arm of N-.The α that obtains, β barrel structure territory (residue 32-335) are overlapped on three dimers of 1,PV8 six aggressiveness continuously to produce Pseudomonas aeruginosa PBGS α, the six aggressiveness set of β bucket.N-terminal arm at the PBGS of people and Pseudomonas aeruginosa does not have significant sequence homogeneity, but conservative α spiral is arranged in the N-terminal arm configuration.Therefore, the comparison of the structure of the eight aggressiveness forms of people PBGS and Pseudomonas aeruginosa PBGS is used to determine the suitable sequence alignment of this alpha helical region section.This information is used to determine the locus of 22-29 amino acids in six aggressiveness of Pseudomonas aeruginosa PBGS.Program Loopy (Xiang, Z., Soto, C.S., and Honig, B. (2002) Proc Natl Acad Sci U SA 99,7432-7437) be used to set up the model of 29-32 amino acids, thereby with the α of N-terminal α spiral and each subunit, β barrel structure territory connects.At last, remaining-terminal amino acid, it is shown in the 1PV8 file, and by the corresponding amino acid whose φ of end user six aggressiveness PBGS, and ω angle information are established on the Pseudomonas aeruginosa PBGS structure.Because the randomness of some N-terminal of people PBGS six aggressiveness (1PV8), the six aggressiveness models of Pseudomonas aeruginosa PBGS lack the 1-9 position residue of subunit A, C and E and the 1-11 position residue of B, D and F.Six aggressiveness Pseudomonas aeruginosa PBGS are with already used disclosed ready-made method (Kundrat before us, L., Martins, J., Stith, L., Dunbrack, R.L., Jr., and Jaffe, E.K. (2003) J Biol Chem 278 31325-31330) sets up six aggressiveness Semen Pisi sativi PBGS model based structures.

Seeking the branch period of the day from 11 p.m. to 1 a.m that preferentially is attached to six aggressiveness PBGS, following discovery is arranged.Analysis to six aggressiveness PBGS shows that " inhibitor " binding site (being also referred to as axillary fossa) of supposition contains the element of three subunit A, B and E.Subunit A and B comprise " the isolating dimer " that has defined, and (subunit A Figure 15) makes the reader directly see and is positioned at α subunit bottom we describe usually herein, the avtive spot of β-bucket central authorities.Subunit B and subunit A share bucket to the bucket interface.Subunit E and subunit B are interact with each other, and the N-terminal arm of one of them subunit is inserted in another subunit α, the bottom of β-bucket.Figure 15 shows the inhibitor-rosmarinic acid described below that docks (docked).In this butt joint result, three subunits that show among rosmarinic acid and the figure all have direct interaction.

Use multiple " micromolecule " molecular library, it is collected by our partner GeorgeMarkham, and the molecule of the PBGS that docking calculation uses commercial docking procedure Glide to try to find out catches six aggressiveness forms.Suppose that power of nature used the morphein catching method, the screening of initial storehouse concentrates on metabolite and natural product.So far ,～1, in the molecular library of 000,000 molecule ,～30, the 000 screened molecule of " axillary fossa " for the six aggressiveness models that can be incorporated into Semen Pisi sativi PBGS.So far, best result is from the natural product rosmarinic acid.

Experimental data from rosmarinic acid

Rosmarinic acid (benzenpropanoic acid,-[[(2E)-3-(3, the 4-dihydroxy phenyl)-and 1-oxygen-2-acrylic] oxygen]-3, the 4-dihydroxy-, (R)-(9CI)) the inhibition data with combine closely slowly that to suppress model consistent, wherein rosmarinic acid preferentially is attached to the Semen Pisi sativi PBGS quarternary structure form less than eight aggressiveness.Figure 16 A, hollow icon shows the specific activity that Semen Pisi sativi PBGS protein concentration relies on, it shows half maximum activity when 3.5 μ g/mlPBGS.This shows at the analysis condition 3.5 μ g/ml places that descend, and the balance of quarternary structure isoform (morpheins) contains the lower isoform of about 50% 8 aggressiveness and about 50% less activity (as, six aggressiveness).If inhibitor works by preferentially being attached to these less forms, can be expected under the condition that the morphein balance contains these less forms has more complete inhibition.In other words, make the protein concentration dependency move to higher protein concentration the expection inhibitor, this is shown as rosmarinic acid (vide infra) in Figure 16 A.Figure 16 B and 16C show to be used to measure how the best confirms the dependent mobile experiment of protein concentration.Figure 16 B shows the dose-effect curve of Semen Pisi sativi PBGS, and it shows before adding substrate makes rosmarinic acid act on albumen in the time of 30 minutes, and the IC50 of this inhibitor is～63 μ M.Show to suppress the dependency to the preincubate time, wherein the inhibition of the rosmarinic acid by arbitrary concentration shows that along with the increase of preincubate time improves rosmarinic acid works as the slow fixation inhibitor.Figure 16 C shows in case inhibition takes place, and then albumen can not recover in 30 minute analysis time.Figure 16 A, the data that 16B and 16C obtain are used to be chosen as and show rosmarinic acid to the necessary appropraite condition of Semen Pisi sativi PBGS protein concentration dependency, and are as follows.The solid rim of Figure 16 A is presented at the protein concentration dependency with 30 μ M rosmarinic acid processing Semen Pisi sativi PBGS specific activity after 30 minutes, obtains half maximum activity at 13.5 μ g/ml PBGS.Therefore, after handling with rosmarinic acid, the balance of quarternary structure form moves to 13.5 μ M from 3.5 μ M; Under these conditions, need 13.5 μ g/ml PBGS to obtain to have the balance of 50% 8 aggressiveness.This is consistent with the explanation that the rosmarinic acid shown in the ball among Figure 10 is stablized less PBGS than the low activity form.Figure 17 supports this conclusion with the native gel electrophoresis data.The 2nd road shows that Semen Pisi sativi PBGS will be separated at least two quarternary structure forms.Swimming on gel with as eight aggressiveness and the equilibrated Semen Pisi sativi PBGS of six aggressiveness consistent (also referring to Fig. 5 B).The 1st and 3 swimming lanes are presented at rosmarinic acid processing back balance and move to less form.The 1st swimming lane shows 250 μ M rosmarinic acid to 30 minutes effect of 142 μ g/ml Semen Pisi sativi PBGS processing, and the 3rd swimming lane shows that interpolation 10mM substrate is to the equilibrated effect of this quarternary structure.

According to our modeling result, mainly by protein protomer A, the hydrogen bond between the polar group of B and E and rosmarinic acid is finished in the interaction of " axillary fossa " of this biphenol compound and Semen Pisi sativi PBGS six aggressiveness.This albumen contains other hydrogen bonding electromotive force in the rosmarinic acid 4.0 dust scopes.Therefore, can prepare rosmarinic acid derivant by the rosmarinic acid molecule is added other hydrogen bonding electromotive force with improved binding ability.For example, can add hydroxyl 5 of arbitrary phenyl to improve to proteic hydrogen bonding.Can obtain by phenyl or the benzyl that replaces 2 of this molecule propanoic acid parts with proteic other hydrophobic interaction.

Although the present invention at length is described with reference to its specific embodiment, it is apparent that those skilled in the art can not depart from the spirit and scope of the invention and it is carried out various changes and modification.

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Sequence table

＜110〉Foxchase Cancer Ct (Fox Chase Cancer Center)

＜120〉six aggressiveness porphobilinogen synzyme are as the target (Hexameric Porphobilinogen Synthase as a Target for the Developmentof Antibiotics and Herbicides) of exploitation antibiotic and herbicide

<130>SCT060059-47

<140>Not Yet Assigned

<141>2004-07-07

<150>60/485,253

<151>2003-07-07

<150>60/577,312

<151>2004-06-04

<160>3

<170>PatentIn version 3.1

<210>1

<211>3142

<212>DNA

<213>Homo sapiens

<400>1

cttacgcggt ctgtgggaga ccggagcggg agacagcggt gacaggagca gcggccggga 60

gcccttaggg aggcagacag agcctgcagc caatgcccca ggagccctcg gttccaacca 120

actgatgccc ctgtgcccac tggcccacgc catgcagccc cagtccgttc tgcacagcgg 180

ctacttccac ccactacttc gggcctggca gacagccacc accaccctca atgcctccaa 240

cctcatctac cccatctttg tcacggatgt tcctgatgac atacagccta tcaccagcct 300

cccaggagtg gccaggtatg gtgtgaagcg gctggaagag atgctgaggc ccttggtgga 360

agagggccta cgctgtgtct tgatctttgg cgtccccagc agagttccca aggacgagcg 420

gggttccgca gctgactccg aggagtcccc agctattgag gcaatccatc tgttgaggaa 480

gaccttcccc aacctcctgg tggcctgtga tgtctgcctg tgtccctaca cctcccatgg 540

tcactgcggg ctcctgagtg aaaacggagc attccgggct gaggagagcc gccagcggct 600

ggctgaggtg gcattggcgt atgccaaggc aggatgtcag gtggtagccc cgtcggacat 660

gatggatgga cgcgtggaag ccatcaaaga ggccctgatg gcacatggac ttggcaacag 720

ggtatcggtg atgagctaca gtgccaaatt tgcttcctgt ttctatggcc ctttccggga 780

tgcagctaag tcaagcccag cttttgggga ccgccgctgc taccagctgc cccctggagc 840

acgaggcctg gctctccgag ctgtggaccg ggatgtacgg gaaggagctg acatgctcat 900

ggtgaagccg ggaatgccct acctggacat cgtgcgggag gtaaaggaca agcaccctga 960

cctccctctc gccgtgtacc acgtctctgg agagtttgcc atgctgtggc atggagccca 1020

ggccggggca tttgatctca aggctgccgt actggaggcc atgactgcct tccgcagagc 1080

aggtgctgac atcatcatca cctactacac accgcagctg ctgcagtggc tgaaggagga 1140

atgatggaga cagtgccagg cccaagaact agaactttaa aacgttcccg gggcctcaga 1200

caagtgaaaa ccaaagtaaa tgctgctttt agaactgtgc cctcatgccc tcttcctgct 1260

cacatgctag cggggcccag cagccctggg tggttttgcc agcatgctaa ctcttgtaac 1320

tcgcagctgc atcctatgag ctctcccaag cttccccgcc cctcccctgg gtcagccgtg 1380

aggcccacct ttgccaccct cagctctttc ctctggtgtg gcttcagctt gaaagcaacc 1440

tggagtcggg ggcacagcct ttggggcctg gctgggagag ggtcttggag cattagggga 1500

agaagagagc agtgggatct tggggcctga gaagccttgg aacgcttctg gcagcagagc 1560

tgggtgtggg aatgaggcct agatcgatat ccctgggtta gagttgaaat ttgccgcaat 1620

tccactggaa ggcatttccc acgaggccag aggttgccag gctgcctgag gtctcctatt 1680

ctactctgaa ccataaaccc agagaagaat tactcattaa ccagcataaa tactgcctga 1740

ggatcaaaac tcagaggcaa agagggagtt cctgactgct agaggtgcca ccaccacaaa 1800

cactttttat tcaggagata ctttttgaga atctctgctc tgttcctagg ttcagtgctg 1860

ggtcctggga atacagcagg acagacctca gcttatctct tcatagaaat tatacaaaga 1920

gaattgggga gacagctaag aagaaaacaa agaaataaag cagttacaaa ttgtgataag 1980

tgctttgaag gaaagaaggg gtctgagaca acaacaggga aggggcctct cttgaaacag 2040

tagttgggaa ggaggcagac atgcaccagt gatgtggtga caggtgctct gaaggaggtc 2100

accaggacct gacctctttg aaggatcaga aaatacttcc ctgaaggact gacatttgag 2160

cctagacctg aagggtgagc catcaagcta agacaattgg ggaagagcat tccagggaga 2220

gggaggagtt gtgcaaaggc cctggggctc cttctagctg gaggaatgca aggctagctt 2280

gtctggagca ctgagaggat ggcctgaact gagtggagag agacagacca ggaccaaacc 2340

atgcagaggt caagggccac attcaccttt tcagagtgac tcaatcaaat ttgtagtttg 2400

taaaagtatt ttaacagctc tgcggcaaag tgcaaatgaa aagtcttgat ggcatggact 2460

ggagcgggga cagtggggat ggagaaaggg gaatggattg tggatgtgtt tagaaggtag 2520

attcgatgtg aaggatgaat ctggcttgac cttctgggtg gctgatgggc catttactga 2580

gatggggcag cctggaagag gaacagaagc agggtcgggg tggagggaga atactaaact 2640

tagcttgaga cattttgcaa taaggaagct atatctagag tgcttatgtg actcacctaa 2700

ggccactcaa caagtttgtg gcagaactgg attagaactg cacagaaaac agccaagctg 2760

ggatttgaac ccatgtagtc caactccaag gcctctgccc ctaaccactg tgccatacca 2820

cctcccaata atcaacagca aaattatagg tctaacaatg ttttatagac acccctccat 2880

ttatgtgatg ggtttgcatc ctgataaacc catcataagt tgaaaatatg atcataagtt 2940

gaaaatatga tcataagtca aaaatgtatt taatatacct aacctaccaa acatcatagc 3000

ttagcctagc ctgccttaaa catgctcaga acacttacat tagcctacag tgggcaaaac 3060

tatccaacac aaaatctata ttgtaataaa gttgtaaaga attttgaata aaaattcaat 3120

atttgaaaaa aaaaaaaaaa aa 3142

<210>2

<211>1154

<212>DNA

<213>Homo sapiens

<400>2

gcagccaaag ccccaggagc cctaggttcc aaccaactga tgcccctgtg cccactggcc 60

cacgccatgc agccccagtc cgttctgcac agcggctact tccacccact acttcgggcc 120

tggcagacag ccaccaccac cctcaatgcc tccaacctca tctaccccat ctttgtcacg 180

gatgttcctg atgacataca gcctatcacc agcctcccag gagtggccag gtatggtgtg 240

aagcggctgg aagagatgct gaggcccttg gtggaagagg gcctacgctg tgtcttgatc 300

tttggcgtcc ccagcagagt tcccaaggac gagcggggtt ccgcagctga ctccgaggag 360

tccccagcta ttgaggcaat ccatctgttg aggaagacct tccccaacct cctggtggcc 420

tgtgatgtct gcctgtgtcc ctacacctcc catggtcact gcgggctcct gagtgaaaac 480

ggagcattcc gggctgagga gagccgccag cggctggctg aggtggcatt ggcgtatgcc 540

aaggcaggat gtcaggtggt agccccgtcg gacatgatgg atggacgcgt ggaagccatc 600

aaagaggccc tgatggcaca tggacttggc aacagggtat cggtgatgag ctacagtgcc 660

aaatttgctt cctgtttcta tggccctttc cgggatgcag ctaagtcaag cccagctttt 720

ggggaccgcc gctgctacca gctgccccct ggagcacgag gcctggctct ccgagctgtg 780

gaccgggatg tacgggaagg agctgacatg ctcatggtga agccgggaat gccctacctg 840

gacatcgtgc gggaggtaaa ggacaagcac cctgacctcc ctctcgccgt gtaccacgtc 900

tctggagagt ttgccatgct gtggcatgga gcccaggccg gggcatttga tctcaaggct 960

gccgtactgg aggccatgac tgccttccgc agagcaggtg ctgacatcat catcacctac 1020

tacacaccgc agctgctgca gtggctgaag gaggaatgat ggaggacagt gccaggccca 1080

agaactagaa ctttcaaacg ttcccggggc ctcagacaag tgacaaccaa agtaaatgct 1140

gcttttagaa ctgt 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 15

Arg Ala Trp Gln Thr Ala Thr Thr Thr Leu Asn Ala Ser Asn Leu Ile

20 25 30

Tyr Pro Ile Phe Val Thr Asp Val Pro Asp Asp Ile Gln Pro Ile Thr

35 40 45

Ser Leu Pro Gly Val Ala Arg Tyr Gly Val Lys Arg Leu Glu Glu Met

50 55 60

Leu Arg Pro Leu Val Glu Glu Gly Leu Arg Cys Val Leu Ile Phe Gly

65 70 75 80

Val Pro Ser Arg Val Pro Lys Asp Glu Arg Gly Ser Ala Ala Asp Ser

85 90 95

Glu Glu Ser Pro Ala Ile Glu Ala Ile His Leu Leu Arg Lys Thr Phe

100 105 110

Pro Asn Leu Leu Val Ala Cys Asp Val Cys Leu Cys Pro Tyr Thr Ser

115 120 125

His Gly His Cys Gly Leu Leu Ser Glu Asn Gly Ala Phe Arg Ala Glu

130 135 140

Glu Ser Arg Gln Arg Leu Ala Glu Val Ala Leu Ala Tyr Ala Lys Ala

145 150 155 160

Gly Cys Gln Val Val Ala Pro Ser Asp Met Met Asp Gly Arg Val Glu

165 170 175

Ala Ile Lys Glu Ala Leu Met Ala His Gly Leu Gly Asn Arg Val Ser

180 185 190

Val Met Ser Tyr Ser Ala Lys Phe Ala Ser Cys Phe Tyr Gly Pro Phe

195 200 205

Arg Asp Ala Ala Lys Ser Ser Pro Ala Phe Gly Asp Arg Arg Cys Tyr

210 215 220

Gln Leu Pro Pro Gly Ala Arg Gly Leu Ala Leu Arg Ala Val Asp Arg

225 230 235 240

Asp Val Arg Glu Gly Ala Asp Met Leu Met Val Lys Pro Gly Met Pro

245 250 255

Tyr Leu Asp Ile Val Arg Glu Val Lys Asp Lys His Pro Asp Leu Pro

260 265 270

Leu Ala Val Tyr His Val Ser Gly Glu Phe Ala Met Leu Trp His Gly

275 280 285

Ala Gln Ala Gly Ala Phe Asp Leu Lys Ala Ala Val Leu Glu Ala Met

290 295 300

Thr Ala Phe Arg Arg Ala Gly Ala Asp Ile Ile Ile Thr Tyr Tyr Thr

305 310 315 320

Pro Gln Leu Leu Gln Trp Leu Lys Glu Glu

325 330

Claims (52)

1. comprise a kind of combination of agents thing, this reagent by being attached to multimeric protein binding site and influence unitary balance thus and influence described multimeric protein, wherein said multimeric protein comprises having a plurality of described unitary set, wherein each described unit comprises first complementary surface and second complementary surface, and one of them unitary first complementary surface and another unitary second complementary surface interrelate, suppose that this set is at least a in the different quarternary structure isoforms, condition is the structure of the described different quarternary structure isoforms of (1) each described unitary structures shape in described multimeric protein, and (2) described unit is in the function of balance and the structure influence multimeric protein of (3) described different quarternary structure isoforms.

2. the compositions of claim 1 wherein influences the formation that described multimeric protein comprises influences the quarternary structure isoform.

3. the compositions of claim 1 wherein influences the function that described multimeric protein comprises influences described multimeric protein.

4. the compositions of claim 3, the function of wherein said multimeric protein be active and influence be suppress or activation at least a.

5. the compositions of claim 4, wherein this reagent is attached at least a in the quarternary structure isoform that has more SA quarternary structure isoform or have greater activity.

6. the compositions of claim 1, wherein each described unit is selected from monomer, dimer, trimer, the tetramer, six aggressiveness and eight aggressiveness.

7. the compositions of claim 1, wherein said multimeric protein is selected from prophobilinogen synzyme and Ia type ribonucleotide reductase.

8. the compositions of claim 7, wherein said multimeric protein is to comprise eight monomeric porphobilinogen synzyme of porphobilinogen synzyme.

9. the compositions of claim 8, wherein this reagent is to be attached to the inhibitor with more SA quarternary structure isoform, and wherein the quarternary structure isoform comprises and is less than eight porphobilinogen synzyme monomers.

10. the compositions of claim 7, wherein said multimeric protein is an Ia type ribonucleotide reductase, and this reagent suppresses Ia type ribonucleotide reductase by being selectively bound to the binding site with more SA quarternary structure isoform uniqueness.

11. compositions, comprise by being attached to and have the monomeric polymer porphobilinogen synzyme than the low activity form of second quantity and suppress to have the inhibitor that the activity form of the monomeric polymer porphobilinogen of first quantity synzyme forms, wherein monomeric first quantity is more than monomeric second quantity.

12. the compositions of claim 11, wherein polymer porphobilinogen synzyme supposes that from antibacterial, archeobacteria or eukaryote eight aggressiveness porphobilinogen synzyme comprise the magnesium binding site of allosteric.

13. the compositions of claim 12, wherein polymer porphobilinogen synzyme comprises catalytic zinc binding site.

14. the compositions of claim 11, wherein polymer porphobilinogen synzyme does not comprise the magnesium binding site and the catalytic zinc binding site of allosteric.

15. the compositions of claim 11, wherein said is six aggressiveness than the low activity form.

16. the compositions of claim 11, wherein said is dimer than the low activity form.

17. the compositions of claim 11, thereby wherein inhibitor substituted metal ion also is combined in metal ion binding site.

18. the compositions of claim 17, wherein metal ion is zinc and/or magnesium.

19. the compositions of claim 11, wherein inhibitor is combined in avtive spot.

20. the compositions of claim 11, wherein inhibitor is not a metal cation.

21. the compositions of claim 11, wherein inhibitor contains the formation that the domain that can not implement to embrace than the low activity form that is less than eight monomeric polymer porphobilinogen synzyme suppresses the activity form of polymer porphobilinogen synzyme by being attached to, and described activity form is eight aggressiveness porphobilinogen synzyme.

22. the compositions of claim 11, wherein inhibitor suppresses the formation of polymer porphobilinogen synthase activity form by being combined in site except that avtive spot and/or metal ion binding site.

23. the compositions of claim 11, wherein inhibitor suppresses the formation of polymer porphobilinogen synthase activity form by the mechanism outside the removal metal ion.

24. the compositions of claim 11 also comprises delivery media, described delivery media is selected from the medicine acceptable medium, oral acceptable medium, the medium of antibacterium medium and effective weeding.

25. the compositions of claim 24, wherein thereby said composition effectively suppresses or prevents the formation and the inhibition of polymer porphobilinogen synthase activity form or prevent antibacterial, archeobacteria and/or Eukaryotic growth or growth, supposes that the activity form of polymer porphobilinogen synzyme contains the magnesium binding site of allosteric.

26. claim 25 compositions, effectively treatment or prevention are by contacting the disease that antibacterial, archeobacteria and/or eukaryote cause.

27. claim 25 compositions, wherein said composition is at least a in medicine, toothpaste, soap, disinfectant, antibiont film composition and the herbicide.

28. the compositions of claim 24, wherein thereby said composition effectively suppresses or prevents the formation and the inhibition of polymer porphobilinogen synthase activity form or prevent antibacterial, archeobacteria and/or Eukaryotic growth or growth, supposes that the activity form of polymer porphobilinogen synzyme does not contain the magnesium binding site and the catalysis zinc of allosteric.

29. the compositions of claim 28, effectively treatment or prevention are by contacting the disease that antibacterial, archeobacteria and/or eukaryote cause.

30. claim 28 compositions, wherein said composition is at least a in medicine, toothpaste, soap and the disinfectant.

31. one kind to being genetically modified herbicide resistant plants to embrace the polymer porphobilinogen synzyme that dimeric polymer exists substantially.

32. the herbicide resistant plants of claim 31, wherein polymer porphobilinogen synzyme is from the people.

33. the herbicide resistant plants of claim 31, wherein polymer porphobilinogen synzyme does not contain the magnesium binding site of allosteric.

34. a compositions contains the inhibitor that is attached to the polymer porphobilinogen synzyme that does not need zinc realization catalysis.

35. a method that influences multimeric protein, this method comprises:

Provide and comprise multimeric protein with a plurality of unit sets, wherein each described unit comprises that first complementary surface is relevant with another unitary second complementary surface with second complementary surface and one of them unitary first complementary surface, suppose that this set is at least a in the different quarternary structure isoforms, condition is the structure of the described different quarternary structure isoform of (1) described unitary structures shape, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quarternary structure isoforms;

The compositions of the claim 1 that comprises reagent is provided, and wherein this reagent influences balance by the binding site that is attached in this set; And

Should gather with reagent and contact, wherein this reagent influences balance by being attached to binding site and influencing described multimeric protein thus.

36. the method for claim 35 wherein influences the formation that described multimeric protein comprises influences the quarternary structure isoform.

37. the method for claim 35 wherein influences the function that described multimeric protein comprises influences described multimeric protein.

38. the method for claim 35, wherein said unit is selected from monomer, dimer, trimer, the tetramer, six aggressiveness and eight aggressiveness.

39. the method for claim 35, the wherein function of the described multimeric protein of this agents influence.

40. the method for claim 39, the function of wherein said multimeric protein be active and influence be suppress or activation at least a.

41. the method for claim 40, wherein this reagent is attached at least a in the quarternary structure isoform that has more SA quarternary structure isoform or have greater activity.

42. the method for claim 41, wherein reagent is attached to the quarternary structure isoform with greater activity.

43. the method for claim 35, wherein said multimeric protein are selected from porphobilinogen synzyme and Ia type ribonucleotide reductase.

44. the method for claim 43, wherein said multimeric protein are to comprise eight monomeric porphobilinogen synzyme of porphobilinogen synzyme.

45. being Ia type ribonucleotide reductase and this reagent, the method for claim 43, wherein said multimeric protein suppress Ia type ribonucleotide reductase by the binding site that is selectively bound to having than low activity quarternary structure isoform uniqueness.

46. adjust the method for cell, tissue or biological metabolically active, this method comprises:

Cell is provided, tissue and organism, cell wherein, tissue or organism comprise and contain the multimeric protein with a plurality of unit sets, wherein each unit comprises that first complementary surface is relevant with another unitary second complementary surface with second complementary surface and unitary first complementary surface, suppose that this set is at least a in the different quarternary structure isoforms, condition is the structure of the described different quarternary structure isoform of (1) described unitary structures shape, and (2) described unit is in the balance and the function of this multimeric protein of structure influence of (3) described different quarternary structure isoforms; And

Provide the compositions of the claim 1 that comprises this reagent to cell, tissue or organism, wherein this reagent influences balance by being attached to the binding site on the unit and influencing the formation of quarternary structure isoform thus and adjust physiologically active.

47. suppress the method that polymer porphobilinogen synzyme forms activity form, this method comprises:

The compositions of claim 11 is applied to polymer porphobilinogen synzyme;

With compositions with get in touch than the low activity form;

Thereby inhibition forms activity form than the set of low activity form and suppresses polymer porphobilinogen synzyme and forms activity form.

48. handle the method for plant growing or growth, comprise that the compositions with claim 27 is administered to plant, wherein this plant is a Herbicid resistant, and to being genetically modified to embrace the polymer porphobilinogen synzyme that dimeric polymer form exists substantially.

49. the method for claim 48, wherein polymer porphobilinogen synzyme does not contain the magnesium binding site of allosteric.

50. prepare the method on antibacterium surface, this method comprises:

The compositions of claim 27 is provided;

Provide the surface to form substrate;

Compositions and surface are formed substrate combine, thus and preparation antibacterium surface.

51. the method for claim 50, wherein the antibacterium surface prevents or suppresses biomembranous formation.

52. the compositions of claim 11, wherein inhibitor is the rosmarinic acid or derivatives thereof.

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