WO2001079457A2 - Crystal structure for rmlc and uses thereof - Google Patents

Abstract

The invention provides the crystal structure of Methanobacterium thermoautotrophicum RmlC (MT RmlC) and identifies the active site thereof. The crystal structure can be used to determine the crystal structure of homologues, analogues, mutants and co-complexes of MT RmlC and to identify and design inhibitors to RmlC. The present invention has applicability in identifying and designing anti-bacterial agents and the treatment of bacterial infections.

Description

Title: Crystal Structure for RmlC and Uses thereof

PRIORITY

This application claims priority from United States Provisional Patent Application No. 60/196,915, filed April 13, 2000, entitled, "Crystal Structure for RmlC and Uses Thereof", the entirety of which is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the crystal structure of dTDP - 4 keto-6- deoxy-D-hexulose, 3, 5-epimerase (RmlC) in the presence and absence of dTDP, a substrate analogue. The invention also relates to methods and uses of the crystal structure in designing and identifying compounds which modulate RmlC activity. In one embodiment the invention relates to inhibitors of RmlC activity.

BACKGROUND OF THE INVENTION

Proteins whose expression and activity are restricted to prokaryotes are attractive antibiotic targets. The comparative analysis of comprehensive genome databases has uncovered a large set of such proteins, which includes enzymes involved in bacterial-specific intermediary metabolism and those involved in the biosynthesis of the bacterial cell wall. The bacterial cell wall comprises a large number of carbohydrates that are not found in mammalian cells, one of which is the activated form of L-rhamnose, dTDP-L-rhamnose. dTDP-L-rhamnose is found in many Gram- negative bacteria. It is converted to L-rhamnose which is found in the O-antigen of many Gram negative bacteria and is a common constituent of cell wall polysaccharides.

The enzymatic mechanism of dTDP-L-rhamnose biosynthesis began to be elucidated more than thirty years ago. dTDP-L-rhamnose is synthesized from α-D-glucose- 1 -phosphate by a set of four bacterial- specific enzymes, called RmlA-D, whose sequences are highly conserved. RmlA, glucose- I -phosphate thymidylyltransferase, catalyzes the synthesis of dTDP-D-glucose from dTTP and α-D-glucose- 1 -phosphate. The next enzyme in the pathway, dTDP-D-glucose 4,6-dehydratase (RmlB) reduces dTDP-D-glucose to dTDP- 4-keto-6-deoxy-D-glucose in an NADH-dependent reaction. RmlC, dTDP-4-keto-6-deoxy-D- hexulose 3,5- epimerase then converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-4- keto-L- rhamnose. Finally, RmlD, dTDP-4-keto-L-rhamnose reductase reduces dTDP-4-keto-L-rhamnose to dTDP-L-rhamnose in an NADPH- dependent reaction (1, 2). More recent studies of dTDP-L-rhamnose biosynthesis have focused on the molecular genetics and structural biology of the corresponding enzymes.

As stated above, dTDP-4~keto-6-deoxy-D-hexulose 3,5- epimerase (RmlC) is involved in the biosynthesis of dTDP-L-rhamnose, which is an essential component of the bacterial cell wall. The amino acid sequence of many RmlC's are known [e.g., Genbank Accession Nos. AAB86256; BAA29502; AF40545; S35299; P26394; AAC70776; P37745; AAB66649; CAB08731; BAA18590; D49906; P37763; P37780; BAA28134; AAC00178; AAD05456; AAD40710; S23343), however, the tertiary or crystal structure of RmlC has not previously been determined. It is the tertiary structure of a protein which provides an understanding of the structural conformational and chemical interactions of the protein and its substrate or other substrates or inhibitors.

There is a need to determine the tertiary structure of RmlC, its structural conformational and chemical interactions. Such information can lead to the development of effective modulators of RmlC activity and more preferably to inhibitors of RmlC activity and antibacterial agents.

SUMMARY OF THE INVENTION

The present inventors have determined the three-dimensional or crystal structure of RmlC in the presence and absence of the ligand, the bound substrate analogue dTDP, and identified the active site thereof. In a further embodiment, the inventors have identified the dTDP and substrate binding sites of RmlC. In yet another embodiment the inventors have identified amino acid residues and regions of the RmlC which are conserved or highly conserved amongst bacterial species and are involved in RmlC activity. In an embodiment the bacteria are any bacteria expressing RmlC, or homologues thereof, preferably Gram negative bacteria, preferably selected from the group consisting of: Methanobacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, Aeitinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseria gonorrhoeae Yersinia enterocolitica, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Klebisiella pneumoniae, Mycobacterium tuberculosis, Serratia marcescens, Burkholderia pseudomallei, Bacteroides fragilis and Salmonella choleraesuis. Most preferably the bacteria is Methanobacterium thermoautotrophicum.

In one embodiment the invention provides a method of using the crystal structure of Methanobacterium thermoautotrophicum RmlC with and/or without bound substrate analogue to solve the structure of a different RmlC crystal or a crystal of a mutant, homologue, analogue, or co-complex of RmlC.

In a further embodiment the invention provides a method of identifying and /or designing modulators of RmlC activity comprising: (a) using the crystal structure of RmlC and the bound substrate analogue to design and select a potential modulator; (b) obtaining said potential modulator, i.e, such as by synthesis or by having it provided from a source;

(c) contacting said potential modulator with RmlC in the presence of a substrate to determine the ability of said potential modulator to modulate RmlC. Preferably, the modulators are inhibitors of RmlC activity.

Preferably the inhibitors are anti-bacterial agents. Preferably, the inhibitors are anti-gram negative bacterial agents. In a preferred embodiment, the RmlC inhibitors of the invention bind to an accessory binding site or an active site of RmlC, but most preferably an active site of RmlC. The active site of RmlC is formed by amino acid residues from β-strands 3 and 4 from one subunit of RmlC with β-strands 5,6,11 & 12 from the other subunit.

In an embodiment, the inhibitor of the invention binds to at least one of the amino residues which form the active site of Methanobacterium thermoautotrophicum RmlC and its homologues: Gln49, Asρ84, Aspl44, Asp 172, Glu31, Lysl71, Glu52, Arg26, Argόl, His64, Hisl20, Cysl35, Ser53, Ser55, Serl69, Asn51, Trpl75, Phe29, Phel22, Tyrl33, Tyrl39. In another embodiment, the RmlC inhibitor binds to at least one of the amino acids which form the dTDP binding site of Methanobacterium thermoautotrophicum RmlC and its homologues which comprises the amino acid residues Phe 29, Tyr 133, Tyr 139, Lys 171, Glu 31, Gin 49, Arg 61 and Arg 26. In another embodiment, the RmlC inhibitor binds to at least one of the amino acids which form the substrate binding site of Methanobacterium thermoautotrophicum RmlC and its homologues which comprises the amino acid residues His 64, His 120, Asp 172, Asp 84 and Lys 73 and more preferably His 64, His 120 and Lys 73. In one embodiment of the invention, the RmlC inhibitor binds to at least one of the amino acid residues 140 to 144 of Methanobacterium thermoautorophicum RmlC and its homologues.

In another embodiment, the RmlC inhibitor binds to amino acid residues V⁵⁹XRGLHZQ⁶⁶ (SEQ ID NO. 18)_' where X is a hydrophobic residue and Z is an aromatic residue, of Methanobacterium thermoautotrophicum RmlC and its homologues.

In yet another embodiment, the inhibitor of the invention binds to an amino acid residue or residues of RmlC which are conserved among all bacteria which express RmlC. In another embodiment, the inhibitor of the invention binds to an amino residue or residues which are conserved among the bacteria selected from the group consisting of: Methano bac teriu m thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, Aeitinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseria gonorrhoeae Yersinia enter ocolitica, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Klebisiella pneumoniae, Mycobacterium tuberculosis, Serratia marcescens, Burkholderia pseudomallei, Bacteroides fragilis and Salmonella choleraesuis. In another embodiment the inhibitor has the three-dimensional structure of the active site of RmlC (e.g an antagonist of the active site), binds dTDP and /or the hexulose substrate, but has no epimerase activity. In a preferred embodiment the inhibitor has the three- dimensional structure of the hexulose substrate binding site of RmlC but not the epimerase activity. In another embodiment, the inhibitor has the three-dimensional structure of both the dTDP and hexulose binding sites of RmlC but does not have epimerase activity. Preferably the RmlC is Methanobacterium thermoautotrophicum RmlC. In a further embodiment, the invention provides a method of designing and synthesizing mutants of RmlC characterized by one or more different properties, as compared to wild-type RmlC. These properties include but are not limited to, altered surface charge, increased /decreased stability to subunit dissociation, latered substrate specificity or higher /lower specific activity. RmlC mutants are useful to identify those amino acid residues that are most important for the enzymatic activity of RmlC. This in turn allows the design of improved inhibitors. RmlC mutants can themselves be RmlC inhibitors.

In one embodiment, the invention provides an anti-bacterial agent which can be used against any bacteria expressing RmlC or its homologues. In one embodiment the bacteria are Archeabacteria or Eubacteria. In another embodiment the anti-bacterial agents of the invention can be a used in industrial and /or therapeutic applications. Industrial applications may include, but are not limited to treating pipelines, other infectable conduits or machinery to prevent or treat such infections.

In a further embodiment, the invention provides a method of treating bacterial infections, by administering to a person in need thereof, an effective amount of an RmlC inhibitor of the invention or designed or identified by a method of the invention. In an embodiment, the bacterial infections are selected from any such infections caused by any bacteria which express RmlC and its homologues, preferably Gram negative bacteria which comprises dTDP-L- rhamnose, or L-rhamnose in its cell wall. In one embodiment the bacteria are selected from the group of bacteria comprising, but not limited to: Methano bacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, Aeitinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseria gonorrhoeae Yersinia enter ocolitica, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Klebisiella pneumoniae, Mycobacterium tuberculosis, Serratia marcescens, Burkholderia pseudomallei, Bacteroides fragilis and Salmonella choleraesuis.

In another embodiment the invention provides a pharmaceutical composition comprising an inhibitor identified or selected by the method of the invention and a pharmaceutically acceptable carrier.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

Figure 1 shows the amino acid sequence of RmlC Methanobacterium thermoautotrophicum (SEQ ID NO. 1) and its alignment with the amino acid sequences of RmlC of other bacteria (SEQ ID NOS. 2- 17). Figure 2A is a ribbon diagram of an Methanobacterium thermoautotrophicum RmlC monomer with a ball and stick model of complexed dTDP. Figure 2B is a stereoview of the C_a trace of a monomer of RmlC.

Figure 3 is ribbon diagram of the Methanobacterium thermoautotrophicum RmlC homodimer with a ball and stick model of complexed dTDP. Figure 3A is a view from the two-fold axis symmetry in the plane. Figure 3B is a rotation of figure 3A by 90° in the plane.

Figure 4 is SigmaA weighted 2Fo-Fc electron density map of the final apo model of RmlC after refinement at 1.5A (Figure 4 A) and of the final dTDP -complexed model after refinement at 1.75 A. Both maps have been contoured at the lσ level.

Figure 5 is a detailed view of the active site of Methanobacterium thermoautotrophicum (MT) RmlC with complexed dTDP. Figure 5A is ribbon diagram showing amino acid residues involved in binding dTDP. Figure 5B is a schematic two-dimensional structure of the active site of RmlC showing amino acid residues and water molecules interacting with complexed dTDP. DETAILED DESCRIPTION OF THE INVENTION The invention is directed to the three-dimensional crystal structure of the apo and a substrate-bound form of the RmlC homologue from Methanobacterium thermoautotrophicum (MT), the use of the structure to solve the structure of RmlC homologues, analogues, and of other crystal forms of RmlC, mutants and co-complexes of RmlC and of the use of RmlC structure and that of its homologues, analogues, mutants and co-complexes to identify the active site and one or more accessory binding sites of RmlC and to design inhibitors of RmlC its homologues, analogues and of other crystal forms of RmlC, mutants and co-complexes of RmlC. The three-dimensional structure of RmlC has uncovered significant structural homology to canavalin A and has provided a model for the mechanism of the dTDP-4-keto-6-deoxy-D-glucose epimerization reaction.

"Homologue" as used herein refers to a protein with at least 30% amino acid sequence identity with MT RmlC or which has at least one functional domain which is characteristic of RmlC.

"Analogue " as used herein refers to a molecule substantially similar in function to either the entire MT RmlC or to a fragment thereof. "Mutant" as used herein refers to an RmlC characterized by replacement or deletion of at least one amino acid from the wild-type RmlC. Such a mutant may, for example, be prepared by site-specific mutagenesis of RmlC cDNA or generated by site-specific incorporation of unnatural or natural amino acids into RmlC using for instance the method of Noren, C. }., et al., Science, 244, pp. 182-188 (1989). (16) "Co-complex " as used herein refers to RmlC, or a mutant or homologue or analogue thereof in covalent or non-covalent association with a chemical entity or compound. dTDP-4-keto-6-deoxy-D-hexulose 3,5-eρimerase (RmlC) is involved in the biosynthesis of dTDP-L-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from

Methanobacterium thermoautotrophicum was determined in the presence and absence of dTDP, a substrate analogue.

The active site is formed by amino acid residues that are conserved in all RmlC sequence homologues. The striking similarity of the architecture of the RmlC active site with that of mandelate racemase suggested a reaction mechanism. The conservation of the active site residues in all RmlC sequence homologues, suggests that the proposed epimerization mechanism is also conserved and that the RmlC structure would be useful in guiding the design of antibacterial drugs.

Amino Acid Abbreviations

A=Ala=Alanine

V=Val=Valine

L=Leu=Leucine I=lle=Isoleucine

P=Pro=Proline

F=Phe=Phenylalanine

W=Trp=Tryptophan

M=Met=Methionine G=Gly=Glydne

S=Ser=Serine

T=Thr=Threonine

C=Cys=Cysteine

Y=Tyr=Tyrosine N=Asn=Asparagine

Q=Gln=Glutamine

D=Asρ=Aspartic Acid

E=Glu=Glutamic Acid

K=Lys=Lysine R=Arg=Arginine H=His=Histidine Crystal Structure of RmlC

The present invention provides crystals of MT RmlC in the presence and absence of dTDP, as well as the tertiary structure of MT RmlC as determined therefrom.

The structure of MT RmlC, as determined by X-ray crystallography data and gel filtration revealed that MT RmlC is a homodimer comprising a central jelly-roll motif, which extends in two directions into longer β-sheets. Binding of the dTDP is stabilized by a combination of ionic interactions with the base and the phosphate, and hydrophobic interactions with the base. The enzyme is symmetrical and has two active sites as determined by dTDP binding and shown in Figure 3. The active sites are located in the central jelly-roll motifs. Each subunit (or monomer) of the homdimer comprises 13 β strands, 1-13, and 3 short α-helices, 1-3, as shown in the ribbon diagram of Figure 2A. An "overview of the structure" of MT RmlC is described below.

The dimer interface of the molecule is formed by an extensive set of hydrophobic and electrostatic contacts between strands: β3A & β5B; β7A &β7B; and αlA & β5B. The designations "A" and "B" as used herein refers to the two subunits or monomers of the homodimer. The designations are used only to indicate interactions between amino acid residues of different subunits but not to refer to one particular subunit. As the RmlC molecule is symmetrical, it should be understood that reference herein to interactions between subunits, such as the dimer interface above, would also include interactions between: β3B & β5A; β7B &β7A; and αlB & β5A. Figures 3A and 3B represent a ribbon drawing of the MT RmlC homodimer.

Determination of the structure of MT RmlC has enabled, for the first time, identification of the active sites and potential accessory binding sites of the enzyme. "Active site" or "active site moiety" as used herein refers to any or all of the following sites: the RmlC substrate binding site (the hexulose), the dTDP binding site, and the site where epimerization occurs. The MT RmlC has two symmetrical active sites. They are formed by β strands 3 and 4 of one subunit and β strands 5, 6, 11, and 12 of the other subunit. The active site is open at the centre of each subunit to permit entry and exit of the ligand through the β face (figures 3A and 5A). The active sites comprise amino acid residues from both subunits. The active site moiety of MT RmlC is characterized by at least amino acid residues: Gin 49, Asp 84, Aspl44, Asp 172, Glu31, Lysl71, Glu52, Arg26, Arg61, His64, Hisl20, Cysl35, Ser53, Ser55, Serl69, Asn51, Trpl75, Phe29, Phel22, Tyrl33, Tyrl39, using the sequence numbering according to Figure 1 (SEQ. ID NO. 1). The amino acid residues involved in substrate binding comprise His 64, His 120, Asp 172, Asp 84 and Lys 73. His 64, His 120 and Lys 73 function as both acid and bases in the epimerization reaction. Amino acid residues involved in dTDP binding include Glu 31, Gin 49, Arg 26, Arg 61, Tyr 133, Tyr 139, Phe 29, and Lys 171.

As can be seen in Figure 1, there are many conserved residues between MT RmlC (SEQ ID NO. 1) and RmlC from other bacteria (SEQ ID NOS. 2-17). Nine charged residues (Arg 26, Glu 31, Arg 61, His 64, Lys 73, Asp 84, His 120, Lys 171, and Asp 172) are located in the proposed active site. Another highly conserved region is V⁵⁹XRGLHZQ⁶⁶ (SEQ ID NO. 18), where X is hydrophobic and Z is aromatic, which forms the base of the active site (where hexulose would be positioned). The site of invariant residues (energetically unfavoured) , such as Gly 62, are also found in many pathogenic bacteria.

Sites other than the active site of RmlC may be important in its activity. "Accessory binding site" as used herein refers to a site of RmlC comprising amino acid residues not necessarily a part of the active site but which may be a locus of RmlC inhibition. Conserved sites located outside the known active site of RmlC (for example as indicated in Figure 1), may be potential accessory binding sites. Differences between the apo and dTDP enzymes may also indicate sites involved in RmlC activity. For instance, amino acid residues 140 to 144 SEQ ID NO. 19) in the presence of the ligand, dTDP, becomes ordered, closing off a portion of the active site and thus may play a role in regulation of the passage of the substrate /product into and out of the active site. Inhibitors directed towards conserved sites or conserved structural regions can be inhibitors for MT RmlC and homologues, analogues and co-complexes thereof. Methods of Identifying and Designing Inhibitors The knowledge obtained concerning MT RmlC may be used to model the tertiary structure of related proteins. It can be used in the determination of the structure of other members of the RmlC family of proteins. The knowledge of the structure of the MT RmlC provides a means of investigating the mechanism of action of these proteins , for example, binding to various receptor molecules. In one embodiment, this can be predicted by various computer models using the structure of RmlC as determined herein as a template. Upon discovering that such binding in fact takes place, the protein structure allows the design and synthesis of small molecules which mimic the functional binding of RmlC to its substrate, and/ or ligand (dTDP) but which lack epimerase activity, and thus inhibit RmlC activity. Alternatively, inhibitors can be designed or identified which bind sites on RmlC which inhibit substrate or ligand binding, or which otherwise inhibit RmlC activity. In another embodiment, inhibitors can be designed by taking the structure of bound substrates, such as the structure of the dTDP substrate analogue and modify it to bind better and tighter.

The following terms are used herein:

The term "naturally occurring amino acids" means the L-isomers of the naturally occurring amino acids. The naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, .gamma.- carboxyglutamic acid, arginine, ornithine and lysine. Unless specifically indicated, all amino acids referred to in this application are in the L-form. The term "unnatural amino acids" means amino acids that are not naturally found in proteins. They can include, but are not limited to modified or derivitized amino acids. Examples of unnatural amino acids used herein, include racemic mixtures of selenocysteine and selenomethionine. In addition, unnatural amino acids include the D or L forms of nor-leucine, para-nitrophenylalanine, homophenylalanine, para- fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine, and D-phenylalanine.

The term "positively charged amino acid" includes any naturally occurring or unnatural amino acid having a positively charged side chain under normal physiological conditions. Examples of positively charged naturally occurring amino acids are arginine, lysine and histidine.

The term "negatively charged amino acid" includes any naturally occurring or unnatural amino acid having a negatively charged side chain under normal physiological conditions. Examples of negatively charged naturally occurring amino acids are aspartic acid and glutamic acid.

The term "hydrophobic amino acid" means any amino acid having an uncharged, nonpolar side chain that is relatively insoluble in water. Examples of naturally occurring hydrophobic amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.

The term "hydrophilic amino acid" means any amino acid having an uncharged, polar side chain that is relatively soluble in water. Examples of naturally occurring hydrophillic amino acids are serine, threonine, tyrosine, asparagine, glutamine, and cysteine.

The term "altered surface charge" means a change in one or more of the charge units of a mutant polypeptide, at physiological pH, as compared to wild-type RmlC. This is preferably achieved by mutation of at least one amino acid of wild-type RmlC to an amino acid comprising a side chain with a different charge at physiological pH than the original wild-type side chain.

The change in surface charge is determined by measuring the isoelectric point (pi) of the polypeptide molecule containing the substituted amino acid and comparing it to the isoelectric point of the wild-type RmlC molecule.

The term "high specific activity" refers to a specific activity of RmlC. This can be monitored by determining second order rate constants or by monitoring epimerization of the substrate. The term "altered substrate specificity" refers to a change in the ability of a mutant or the effect of an inhibitor of RmlC on wild-type RmlC activity. Substrate specificity may be measured by substrate assays, such as fluorogenic assays, HPLC, ELISA or other assays or monitoring systems known in the art. In one embodiment, RmlC activity is measured by monitoring the RmlD activity. RmlD converts the product of RmlC, dTDP-4-keto-mannose to dTDP-rhamnose, by the oxidation of NAD(P)H. Oxidation of NAD(P)H is monitored by a decrease in absorbance at 340 ran. The "kinetic form" of RmlC refers to the condition of the enzyme in its free or unbound form or bound to a chemical entity at either its active site or accessory binding site.

A "competitive" inhibitor is one that inhibits RmlC activity by binding to the same kinetic form, of RmlC, as its substrate binds—thus directly competing with the substrate for the active site of RmlC. Competitive inhibition can be reversed completely by increasing the substrate concentration.

An "uncompetitive" inhibitor is one that inhibits RmlC by binding to a different kinetic form of the enzyme than does the substrate. Such inhibitors bind to RmlC already bound with the substrate and not to the free enzyme. Uncompetitive inhibition cannot be reversed completely by increasing the substrate concentration.

A "non-competitive" inhibitor is one that can bind to either the free or substrate bound form of RmlC. Those of skill in the art may identify inhibitors as competitive, uncompetitive or non-competitive, by computer fitting enzyme kinetic data using standard equations according to Segel, I. H., Enzyme Kinetics, J. Wiley & Sons, (1975) (17). It should also be understood that uncompetitive or non-competitive inhibitors according to this invention may bind to the accessory binding site. The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and an RmlC molecule or portions thereof. The association may be non- covalent— wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions—or it may be covalent.

The term "structure coordinates" refers to mathematical coordinates derived from mathematical equations known in the art related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of an RmlC molecule in crystal form (see Protein Data Bank at http://www.rcsb.org, where PDB ID No. 1EPZ depicts the structural co-ordinate data determined by the present inventors, of the Methanobacterium thermoautotrophicum (MT) RmlC molecule in crystal form with dTDP and PDB ID No. 1EP0 depicts it in apo form. These strutural coordinate data are incorporated in the entirety herein by reference.). The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.

The term "heavy atom derivatization" refers to the method of producing a chemically modified form of a crystal of RmlC. In practice, a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thimerosal or uranyl acetate, which can diffuse through the crystal and bind to the surface of the protein. The location(s) of the bound heavy metal atom(s) can be determined by X-ray diffraction analysis of the soaked crystal. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the enzyme. Blundel, T. L. and N. L. Johnson, Protein Crystallography, Academic Press (1976) (18). Those of skill in the art understand that a set of structure coordinates determined by X-ray crystallography is not without standard error. For the purpose of this invention, any set of structure coordinates for RmlC or RmlC homologues, analogues or mutants that have a root mean square deviation of protein backbone atoms (N, C.alpha., C and 0) of less than 0.75.ANG. when superimposed— using backbone atoms—on the structure coordinates listed in Protein Data Bank PDB ID Nos 1EP0 and 1EPZ shall be considered identical. The term "unit cell" refers to a basic parallelepiped shaped block. The entire volume of a crystal may be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal. The term "space group" refers to the arrangement of symmetry elements of a crystal.

The term "molecular replacement" refers to a method that involves generating a-preliminary model of an RmlC crystal whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal. Lattman, E., "Use of the Rotation and Translation Functions", in Methods in Enzymology, 115, pp. 55-77 (1985) (19); M. G. Rossmann, ed., "The Molecular Replacement Method", Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York, (1972) (20). Using the structure coordinates of MT RmlC provided by this invention, molecular replacement may be used to determine the structure coordinates of a crystalline mutant or homologue of MT RmlC or of a different crystal form of RmlC.

For the first time, the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including inhibitory compounds, capable of binding to the active site or accessory binding site of RmlC, in whole or in part. The present invention further permits the design and selection of chemical entities and compounds, which can mimic the substrate and /or ligand binding site but which lack epimerase activity, thus inhibiting RmlC activity.

One approach enabled by this invention, is to use the structure coordinates of RmlC (Protein Data Bank PDB ID Nos 1EPZ and/or 1EP0) to design compounds that bind to the enzyme and alter the physical properties of the compounds in different ways, e.g., solubility. For example, this invention enables the design of compounds that act as competitive inhibitors of the RmlC enzyme by binding to, all or a portion of, the active site of RmlC. This invention also enables the design of compounds that act as uncompetitive inhibitors of the RmlC enzyme. These inhibitors may bind to, all or a portion of, the accessory binding site of an RmlC already bound to its substrate and may be more potent and less non-specific than known competitive inhibitors that compete only for the RmlC active site. Similarly, non-competitive inhibitors that bind to and inhibit RmlC whether or not it is bound to another chemical entity may be designed using the structure coordinates of RmlC of this invention. Inhibitors of the invention can be organic compounds, proteins or non-organic compounds.

A second design approach is to probe an RmlC crystal with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate RmlC inhibitors and the enzyme. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of where each type of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their RmlC inhibitor activity. Travis, ]., Science, 262, p. 1374 (1993).(21)

This invention also enables the development of compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to RmlC, with RmlC. Thus, the time-dependent analysis of structural changes in RmlC during its interaction with other molecules is enabled.

The reaction intermediates of RmlC can also be deduced from the reaction product in co-complex with RmlC. Such information is useful to design improved analogues of known RmlC inhibitors or to design novel classes of inhibitors based on the reaction intermediates of RmlC and RmlC-inhibitor co-complex. This provides a novel route for designing RmlC inhibitors with both high specificity and stability. Another approach made possible and enabled by this invention, is to screen computationally small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to the RmlC enzyme. In this screening, the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy. Meng, E. C. et al, J. Comp. Chem., 13, pp. 505-524 (1992).(22)

Because RmlC may crystallize in more than one crystal form, the structure coordinates of RmlC, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of RmlC. They may also be used to solve the structure of RmlC mutants, RmlC co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of RmlC. One method that may be employed for this purpose is molecular replacement. In this method, the unknown crystal structure, whether it is another crystal form of RmlC, an RmlC mutant, or an RmlC co-complex, or the crystal of some other protein with significant amino acid sequence homology to any functional domain of RmlC, may be determined using the RmlC structure coordinates of this invention as provided in Protein Data Bank, PDB ID Nos. 1EPZ and/or 1EP0 (http://www.rcsb.org). This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio. In addition, in accordance with this invention, RmlC mutants may be crystallized in co-complex with known RmlC inhibitors. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of wild-type RmlC. Potential sites for modification within the various binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between RmlC and a chemical entity or compound. All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 2- 3.ANG. resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, ©1992, distributed by Molecular Simulations, Inc.). See, e.g., Blundel & Johnson, supra; Methods in Enzymology, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985) (23). This information may thus be used to optimize known classes of RmlC inhibitors, and more importantly, to design and synthesize novel classes of RmlC inhibitors. The structure coordinates of RmlC mutants provided in this invention also facilitate the identification of related proteins or enzymes analogous to RmlC in function, structure or both, thereby further leading to novel therapeutic modes for treating bacterial infections.

The design of compounds that bind to or inhibit RmlC according to this invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with RmlC, its substrate and/or ligand. Second, the compound must be able to assume a conformation that allows it to associate with RmlC, its substrate and/or ligand. Although certain portions of the compound will not directly participate in this association with RmlC, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., active site or accessory binding site of RmlC, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with RmlC.

The potential inhibitory or binding effect of a chemical compound on RmlC may be analyzed prior to its actual synthesis and testing by the use of computer modelling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and RmlC, its substrate or ligand, synthesis and testing of the compound is obviated. However, if computer modelling indicates a strong interaction, the molecule may then be synthesized ( or otherwise obtained if not novel) and tested for its ability to bind to RmlC, its substrate and /or ligand using assays known in the art, such as fluorescent substrate assays and HPLC. In one embodiment, RmlC activity is measured by monitoring the RmlD activity. RmlD converts the product of RmlC, dTDP-4-keto-mannose to dTDP-rhamnose, the oxidation of NAD(P)H. Oxidation of NAD(P)H is monitored by a decrease in absorbance at 340 nm. In this manner, synthesis of inoperative compounds may be avoided.

An inhibitory or other binding compound of RmlC may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding sites or other areas of RmlC.

One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with RmlC and more particularly with the binding sites of the RmlC active site or accessory binding site. This process may begin by visual inspection of, for example, the active site on the computer screen based on the RmlC structural coordinates. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within an individual binding site of RmlC. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER. Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include, but are not limited to:

1. GRID (Goodford, P. ]., "A Computational Procedure for

Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 28, pp. 849-857 (1985)) (24) . GRID is available from Oxford

University, Oxford, UK. 2. MCSS (Miranker, A. and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins: Structure. Function and Genetics, 11, pp. 29-34 (1991)) (25). MCSS is available from Molecular Simulations, Burlington, Mass.

3. AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure. Function, and Genetics, 8, pp. 195-202 (1990)) (26). AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.

4. DOCK (Kuntz, I. D. et al., "A Geometric Approach to Macromolecule-Ligand Interactions", J. Mol. Biol., 161, pp. 269-288 (1982)) (27). DOCK is available from University of California, San Francisco, Calif.

Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or inhibitor. Assembly may be proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of RmlC. This would be followed by manual model building using software such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include but are not limited to:

1. CAVEAT (Bartlett, P. A. et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active

Molecules". In "Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989))(28). CAVEAT is available from the University of California, Berkeley, Calif. 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Martin, Y. C, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992)) (29).

3. HOOK (available from Molecular Simulations, Burlington,

Mass.).

Instead of proceeding to build an RmlC inhibitor in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other RMLC binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion(s) of a known inhibitor(s).

These methods include but are not limited to:

1. LUDI (Bohm, H.-J., "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)) (30). LUDI is available from Biosym Technologies, San Diego, Calif.

2. LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991)) (31) LEGEND is available from Molecular Simulations, Burlington, Mass.

3. LeapFrog (available from Tripos Associates, St. Louis, Mo.). Other molecular modelling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al., "Molecular Modeling Software and Methods for Medicinal Chemistry", J. Med. Chem., 33, pp.

883-894 (1990) (32) See also, Navia, M. A. and M. A. Murcko,

"The Use of Structural Information in Drug Design", Current

Opinions in Structural Biology, 2, pp. 202-210 (1992). (33)

Once a compound has been designed or selected by the above methods, the efficiency with which that compound may bind to RmlC may be tested and optimized by computational evaluation. For example, a compound that has been designed or selected to function as an RmlC- inhibitor must also preferably traverse a volume not overlapping that occupied by the active site when it is bound to the native substrate. An effective RmlC inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient RmlC inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, preferably, not greater than 7 kcal/mole. RmlC inhibitors may interact with the enzyme in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the inhibitor binds to the enzyme.

A compound designed or selected as binding to RmlC may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the inhibitor and the enzyme when the inhibitor is bound to RmlC, preferably make a neutral or favorable contribution to the enthalpy of binding.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include: Gaussian 92, revision C [M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. ©1992]; AMBER, version 4.0 [P. A. Kollman, University of California at San Francisco, ©1994]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. ©1994]; and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif. ©1994). These programs may be implemented, for instance, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known to those skilled in the art.

Once an RmlC-binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to RmlC by the same computer methods described in detail, above.

The present invention also enables mutants of RmlC and the solving of their crystal structure. More particularly, by virtue of the present invention, the location of the active site, accessory binding site and interface of RmlC based on its crystal structure permits the identification of desirable sites for mutation. Mutants of RmlC can also be designed to competively compete with wild-type RmlC in substrate/ligand binding, or otherwise act as inhibitors of RmlC. For example, mutation may be directed to a particular site or combination of sites of wild-type RmlC, i.e., the accessory binding site or only the active site, or a location on the interface site may be chosen for mutagenesis. Similarly, a location on, at or near the enzyme surface may be replaced, resulting in an altered surface charge of one or more charge units, as compared to the wild-type enzyme. Alternatively, an amino acid residue in RMLC may be chosen for replacement based on its hydrophillic or hydrophobic characteristics.

Such mutants may be characterized by any one of several different properties as compared with wild-type RmlC. For example, such mutants may have altered surface charge of one or more charge units, or have an increased stability to subunit dissociation. Or such mutants may have an altered substrate specificity in comparison with, or a higher specific activity than, wild-type RmlC.

Once the RmlC mutants have been generated in the desired location, i.e., active site or accessory binding site, the mutants may be tested for any one of several properties of interest. For example, mutants may be screened for an altered charge at physiological pH. This is determined by measuring the mutant RmlC isoelectric point (pi) in comparison with that of the wild-type parent. Isoelectric point may be measured by gel-electrophoresis according to the method of . Wellner, D., Analyt. Chem., 43, p. 597 (1971) (34) A mutant with an altered surface charge is an RmlC polypeptide containing a replacement amino acid located at the surface of the enzyme, as provided by the structural information of this invention, and an altered pi.

The mutants of RmlC or other identified or designed protein inhibitors of RmlC may be prepared in a number of ways known in the art, such as using recombinant DNA methods, such as by incorporating the cDNA encoding the protein in a suitable expression vector, and host cell, expressing the protein under desirable conditions, and if necessary, harvesting or isolating the protein from the expression system.

It should be understood that not all expression vectors and expression systems function in the same way to express DNA sequences of this invention . Neither do all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, an important consideration in selecting a vector, will be the ability of the vector to replicate in a given host. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.

In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability, its compatibility with the DNA sequence encoding the protein to be expressed, particularly with regard to potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the expressed protein to them, their ability to secrete mature products, their ability to fold proteins correctly, their fermentation requirements, the ease of the purification of the protein and safety. Within these parameters, one of skill in the art may select various vector /expression control system/host combinations that will produce useful amounts of the protein to be expressed. The protein produced in these systems may be purified by a variety of conventional steps and strategies.,

For mutations, the wild-type sequence of RMLC may be mutated in those sites identified using this invention as desirable for mutation, by means of oligonucleotide-directed mutagenesis or other conventional methods.

For methods of carrying out the above procedures, see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989). (35) Alternatively, mutants of-RmlC may be generated by the site specific replacement of a particular amino acid with an unnaturally occurring amino acid. In addition, RmlC mutants may be generated through replacement of an amino acid residue, or a particular cysteine or methionine residue, with selenocysteine or selenomethionine. This may be achieved by growing a host organism capable of expressing either the wild-type or mutant polypeptide on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both).

The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149- 2154) (36) or synthesis in homogenous solution (Houbenwyl, 1987, Methods of Organic Chemistry, ed. E. Wansch Vol. 15 I and II, Thieme, Struttgart) (37). Industrial and Therapeutic Applications

The crystal structure of RmlC can be used to identify and design modulators of RmlC. Modulators of RmlC can either increase or decrease RmlC activity. In a preferred embodiment, the crystal structure of RmlC can be used to identify and design inhibitors of RmlC, its homologues, analogues, mutants and co-complexes thereof. Such inhibitors can be used as anti-bacterial agents (i.e., antibiotics). The antibiotics can be used to treat or prevent bacterial infections which are caused by bacteria which express RmlC. They are preferably used to treat Gram-negative bacterial infections, such as, but not limited to infections caused by the following bacteria: Methanobacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestήs, Pyrococcus hoikoshii, Aeitinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseria gonorrhoeae Yersinia enterocoUtica, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Klebisiella pneumoniae, Mycobacterium tuberculosis, Serratia marcescens, Burkholderia pseudomallei, Bacteroides fragϊlis and Salmonella choleraesuis.

The anti-bacterial agents of the invention can be used in industrial and therapeutic applications. Examples of industrial applications include but are not limited to preventing or treating bacterial infections in piplines or other conduit or machinery, or items susceptable to infection. Thus the present invention encompasses a method of treating or preventing such bacterial infections, preferably by applying an effective amount of the inhibitor. The invention also comprises anti- bacterial compositions comprising the inhibitor.

Further, the anti-bacterial agents of the invention can be used to treat any animal of the animal kingdom, including humans, which is susceptible to such bacterial infections. "Individual" and 'patient" as used herein refers to such an animal. As such the present invention encompasses within its scope a method of treating a bacterial infection in an individual by administering an effective amount said inhibitor in a biologically compatible form suitable for administration in vivo to the individual in need thereof. As used herein "biologically compatible form suitable for adminstration in vivo" means a form of the substance to be administered in which therapeutic effects outweigh any toxic effects.

An effective amount of the inhibitor or pharmaceutical compositions of the invention is defined as an amount of the inhibitor or pharmaceutical composition, at dosages and for periods of time necessary to achieve the desired result. For instance, an effective amount or therapeutically active amount of a substance may vary according to factors such as disease state, age, sex, and weight of the recipient and route of adminstration. Any suitable route of administration may be employed for providing the animal with an effective amount of the inhibitor Dosage regima may be adjusted to provide an optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Pharmaceutical Compositions

The present invention is also directed to pharmaceutical compositions comprising inhibitors of or a pharmaceutically acceptable salts thereof , as an active ingredient. "Pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic and organic bases. The pharmaceutical compositions of the invention may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The pharmaceutical compositions of the invention can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to animals. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA 1985). (38) On this basis the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

The preparation and formulations of the pharmaceutical compositions will depend on the desired form of administration. The pharmaceutical compositions of the invention can be formulated to be administered by a variety of routes, such as by injection (subcutaneous, intravenous, intramuscular, etc..) oral administration, inhalation or topical (i.e., transdermal or rectal application). The pharmaceutical composition can be prepared in a variety of forms, such as, tablets, troches, dispersions, suspensions, solutions, powders, capsules, creams, ointments, aerosols, and the like. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound, and/or to ensure delivery of the active substance at a particular site of the body. The amount of active substance per unit form of the pharmaceutical composition may vary depending on the form of the composition and optimum dosage regime. The following non-limiting examples are illustrative of the present invention:

EXAMPLES Protein Expression and Purification

The RmlC gene from MT genomic DNA was amplified by PCR and cloned into the pet 15b (Novagen) expression vector. Recombinant dTDP-4-keto-6-deoxy-D-hexulose epimerase (RmlC) was expressed in Esherichia coli BL21 Gold (DE3) cells (Stratagene) harboring a plasmid encoding three rare E.coli tRNA genes (AGG and AGA for Arg and ATA for He). Conditions for protein expression and purification were similar to those in the Qiagen protein purification handbook (Q1A expression TM, A Handbook for High-Level Expression and Purification of 6xHis-Tagged Proteins, March 1999, QUIAGEN (39)) except that a heat step (55°C for 10 min) and a centrifugation step were introduced after cell lysis to remove most contaminating E. coli proteins. Purified RmlC was dialyzed against 10 mM HEPES and 500 mM NaCl and concentrated to 10 mg/ml using BioMax concentrators (Millipore). For the preparation of selenomethionine (Se-Met) protein, RmlC was expressed in a methionine auxotroph strain B834(DE3) (Novagen) and purified under the same conditions as native RmlC with the addition of 5 mM β-mercaptoethanol in all of the buffers. Gel Filtration

Gel filtration of RmlC was performed with a Superdex 200 prep 16/60 (Pharmacia) column equilibrated with 10 mM HEPES and 500 mM NaCl using an HPLC (LKB-Wallac). Protein standards included aldolase, bovine serum albumin, ovalbumin and cytochrome c. Chromatography was performed at 4°C at a flow rate of 0.5 ml/min. Crystallization

An initial crystallization condition was obtained using a sparse crystallization matrix (Hampton Research Crystal Screen™ I) using hanging drop vapor diffusion. This condition was modified slightly by varying the pH and concentration of polyethylene glycol and yielded crystals suitable for native and MAD data collection. Crystals suitable for data collection grew in 10% PEG 4000 and 100 mM sodium acetate at pH 4.6 in 2 to 4 days at 22°C using hanging drops (3μL: 3 μL protein:precipitant ratio). Monoclinic crystals grew to approximate dimensions of 600 x 200 x 200 microns³. These crystals belonged to the C2 space group with unit cell dimensions 67.7 X 53.1 X 51.7 A and β = 96.6°. There was a single molecule in the asymmetric unit with a Matthews coefficient = 2.3 A³ / Dalton and an estimated solvent content of 46%. Soaking of depimerase crystals was carried out in lOmM dTDP with 10% PEG 4000 and lOOmM sodium acetate at pH 4.6 for 4 Hours. X-Ray Diffraction and Structure Determination

The structure of RmlC was determined by the multiwavelength anomalous dispersion (MAD) method using selenium as the anomalous scatterer. A three-wavelength MAD experiment was performed at the BioCARS 14BMD beamline at the Advanced Photon Source. The high- resolution data of the native crystal were collected with the BioCARS 14BMD beamline. The MAD and native data were processed and scaled with the Denzo/Scalepack (3) suite of programs. Three selenium sites were located using SOLVE (4) and refined using PHASES (5). Solvent flattening was done using PHASES. Model building was done with 0 (6). CNS (7) was used for refinement with multiple rounds of minimization, simulated annealing, B-group, and individual B-factor refinement followed by manual rebuilding. Most of the water molecules were picked using CNS and additional ones were manually added after manually verification using O. The crystallographic data collection and refinement statistics are given in Tables 1 and 2 and Protein Data Bank PDB ID Nos. 1EPZ and 1EP0 (http://www.rcsb.org).

RESULTS AND DISCUSSION

Structure Determination

The structure of selenomethionine-enriched RmlC was determined by the multiwavelength anomalous dispersion (MAD) method and refined against 1.5 A resolution data to a working R-f actor of 0.183 and a free R-factor of 0.211. The refined apo model contains 183 amino acids (residues 3-185) and 127 water molecules (Table 2 and Figure 2). The electron density of the apo form, which was used to build the model, is of excellent quality except for the loop between residues 141 and 143. The dTDP complex model was refined against 1.75 A resolution data to a working R-factor of 0.195 and a free R-factor of 0.224. This model contains 183 amino acid residues, 119 water molecules and one molecule of dTDP (Table 2, Figure 2). The first two amino acids at the N- terminus are not visible in the electron density map in either model. PROCHECK (8) was used to evaluate the stereochemistry of both of the refined models which showed that more than 90% of the residues are in the allowed region and only one amino acid (Glu 68) was in the disallowed regions because it is present in a gamma turn between β6 and β-.7.

Overview of the Structure

RmlC is a homodimer; this was confirmed by gel filtration analysis (data not shown). The momomer comprises thirteen β-strands and three short -helices (Figure 2A). The strands and helices comprise the following amino acid residues: βl(3-10);β2(13-20);β3(21-25);β4(26- 30);β5(48-55);β6(59-65);β7(72-79);β8(81-88);β9(99-105);βl0 (109- 115);βll(118-125);βl2(129-136);βl3(143-148);αl(35-42);α2(169-175);α3(177- 182).

Eight of the β-strands are arranged in a central 8-stranded antiparallel β-sheet (strands β5A to βl2A) that resembles a jelly-roll (Figure 2). Four other strands βlA, β2A, β3B and β4B (from subunits A and B) extend from strands β5A, β7A, βlOA and βllA from the jelly-roll to form an 8-stranded anti-parallel P-sheet. A second β-sheet is formed by β-13A aligned in an antiparallel manner with strands β6A, β8A, B9A, and βllA (Figure 3). The helices are found on the periphery of the molecule. Helix, αl packs against strand βl from terminal β-sheet. Helices, α2 and α3, flank the carboxy terminus of the subunit and are also involved in important crystal packing interactions. Helix, al, also contributes to the active site of the same subunit.

The dimer interface is formed by an extensive set of hydrophobic and electrostatic contacts between β3A & β5B, βTA & β7B and αlA & β5B. Some of these ionic interactions include R61 to D24 via a water molecule and the formation of two salt bridges (E52 to R76 and D50 to K134). Hydrophobic interactions occur between residues Phe 33 Ala 36, Tyr28, Arg 26 (aliphatic side chain), Val 48, Val 59, lie 78 and Leu 138 at the subunit interface. These interactions result in a total buried surface area of 3042 A² out of a 16306 A² for the dimer.

A search for structural homologues using the program Dali (9) revealed that RmlC is homologous to canavalin A, phosphomannose isomerase and arabinose operon regulatory protein (Ara C). The nearest structural neighbour is canavalin A, which has a Z-score of 6.4 and root mean square deviation (RMSD) of l.δA over 87 out of 178 C_α, atoms. The overall core topology of these molecules is similar to the jelly-roll structural motif. Location of the Active Site

The residues involved in substrate binding and catalysis were identified by determining the structure in the presence of a substrate analog, dTDP. The electron density map of the complex revealed a well- ordered dTDP with high occupancy (Figure 4). The substrate-binding site is located in the center of a cavity formed by the jelly-roll structural motif (which is at, the middle of one face of a subunit of RmlC, Figure 3). Residues from β-strands 3 & 4 from one subunit combine with β-strands 5, 6 11 & 12 from the other subunit to form a complete active site. The homodimer enzyme is symmetrical and thus each enzyme has two active sites. The active site is open at the center of each subunit to permit entry and exit of the ligand through the B-face (Figure 3). The active site is lined with a number of charged residues Q49, D84, D144, D172, E31, K73, K171, E52, R26, R61, H64, H120 and C135) and a number of H-bonding residues (S53, S55, S169, Q49, N51) which comprise a network of ionic and H- bonding interactions for substrate binding and catalysis. The active site is also lined with aromatic residues (W175, F29, F122, Y133 and Y139), which provide favorable environments for the base moiety of dTDP and potentially for the sugar moiety of the substrate (Figure 5).

Comparison Between Apo and dTDP Bound dTDP-4-keto-6-deoxy-D- hexulose Epimerase The structure of a subunit of the apo form of RmlC is very similar to that of the dTDP-bound enzyme with an overall root mean square deviation (rmsd) of 0.33 A for 183 C_α atoms. There are, however, some notable differences between the apo and dTDP enzymes. The most prominent differences occur within residues 140 to 144 (SEQ. ID. NO. 19) which are visible in the presence of dTDP. In the presence of the ligand, this loop becomes ordered, closing off a portion of the active site. This loop may be important in regulating the passage of the substrate /product into and out of the active site and may serve to keep the external solvent molecules away from the active site.

Model For Substrate Binding

The dTDP portion dTDP-4-keto-6-deoxy-D-hexulose anchors the substrate in the active site of the enzyme. dTDP binds between strands β5, β6, βll & βl2 of one subunit and β3 & β4 of the other subunit. Aromatic stacking is observed between Tyr 139 & Phe 29 and the base of dTDP. In fact, the electron density of the side chains of Tyr 133, Tyr 139 and Lys 171 was observed only in the presence of dTDP. Tyr 139 stacks against the base moiety of dTDP and Lys 171 makes ionic interactions with an oxygen on the β-phosphate of dTDP through a water molecule. The base of dTDP is bound in an anti-conformation relative to the ribose ring (Figure 5) by hydrogen bonding to Glu 31B and Gin 49A. The diphosphate portion of dTDP is securely anchored to the protein by ionic interactions between the oxygens on the phosphates with Arg 61A and Arg 26B. In addition to these interactions, there are also a number of H- bonds between the phosphate oxygens and the enzyme via water molecules (waters 1035, 1036, 1071 and 1095). Model For Enzymatic Mechanism

The reactive center(s) for the epimerization of hexulose by RmlC were determined by applying distance constraint based on existing mechanisms of epimerization (10, 11). Sugar phosphate epimerization centers are commonly about 5 to 7A away from the phosphorous atom of the β-phosphate (11). Within H-bonding distances from the epimerization centers, a number of ionizable groups (His 64, His 120, Asp 172, Asp 84 and Lys 73) that participate in acid/base chemistry were identified. Based on the distance restraints His 64, His 120 and Lys73 function as both acids and bases in the epimerization reaction. Both His 64 and His 120 are strategically placed in the active site such that they are within H-bonding distance from the epimerization sites of the hexulose moiety of the substrate. Interestingly, the ε-imine of His 64 is H-bonded to one of the carboxylates of Asp 172 and similarly for His 120 with Asp 84. Interactions between His and Asp residues of this nature were observed in the active site of mandelate racemase (MR) where they functioned as catalytic dyads in the acid /based mechanism (12). Analogous to mandelate racemase, aspartate negates the acid /base character of the imidazolium group of the histidine such that the histidine is suitably charged to abstract a proton from the α-position of hexulose. Lys 73 is within interacting distance from the hexulose moiety of the substrate. This Lys residue demonstrate both acid /base characteristics in the epimerization reaction and may also play a role in stabilizing the enolic intermediate of hexulose. There are also a number of well- ordered water molecules occupying this region of the active site and are within H- bonding distance to the hexulose moiety of the substrate. These water molecules are involved in proton exchange with acidic groups in the active site and may even participate in proton transfer to the enolate intermediate of hexulose. Conservation of Function

The tertiary structure of Methanobacterium thermoautorophicum RmlC can be used to identify structural conformational and chemical interactions of the protein (and homolgues, and mutants thereof) and its substrate. It can also be used to identify functional sites in RmlC of other bacterium and in homologues and analogues thereof

To examine the generality of the reaction mechanism, it was examined if the residues determined to be important for binding and catalysis were conserved. The sequences of seventeen randomly selected members of the RmlC family were aligned. Thirty-two residues were conserved in all sequences (Figure 1, SEQ. ID. NOS. 1-17) of which nine charged residues (Arg 26, Glu 31, Arg 61, His 64, Lys 73, Asp 84, His 120 and Lys 171, Asp 172) are located in the active site. Another highly conserved region, which forms strand β6 (residues V⁵⁹XRGLHZQ⁶⁶ where X is hydrophobic and Z is aromatic SEQ . ID. NO. 18), forms the base of the active site (where hexulose would be positioned in the reaction). Two of the residues in strand β6, Arg 61 and His 64 are involved in substrate-binding and the catalytic reaction of hexulose epimerization respectively. There is also an invariant glycine in this region. This glycine forms a cis- peptide (Gly 62). Since this is an energetically unfavored conformation; this alternate structure might explain the importance of this invariant residue in the active site. Notably, the set of invariant residues are found in the sequences of RmlC homologues from many pathogenic bacteria, suggesting that the architecture of the active site is also conserved and that this structure can be used to design and evaluate modulators of RmlC acitivty and in the development of antibacterial drugs.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. TABLE 1

TABLE 2

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

1. Graninger, M., Nidetzky, B., Heinrichs, D.E., Whitfield, C, and Messner, P. (1999). / Biol Chem. 274, 25069 - 25077

2. Stern, R.J., Lee, T.Y., Lee, T.J., Yan, W., Scherman, M.S., Vissa, V.D., Kim, S.K., Wanner, B.L., and McNeil, M.R. (1999) Microbiology. 145, 663-671

3. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307- 326

4. Terwilliger, T.C., and Berendzen, J. (1999) Acta Crystallogr. D 55, 849-61

5. Furey, W., and Swaminathan, S., (1997) Methods Enzymo. 277,

6. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991) Acta Crystallogr. A 47, 110-119 ^"

7. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T., and Warren, G.L., (1998) Acta Crystallogr. D 54, 905-921

8. Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Thornton, J.M. (1993) /. Appl. Crystallogr., 283-291

9. Holm, L., and Sander, C. (1993) /. Mol. Biol. 233, 123-138

10. Thoden, J.B., Hegeman, A.D., Wesenberg, G., Chapeau, M.C., Frey, P.A., and Holden, H.M., (1997) Biochemistry. 36,6294-6304 11. Kopp, J., Kopriva ,S., Suss, K.H., and Schulz, G.E ., (1999) JMol Biol. 287, 761-71

12. Schafer, S.L., Barrett, W.C., Kallarakal, A.T., Mitra, B., Kozarich, J.W., Gerlt, J.A., Clifton, J.G., Petsko, G.A., and Kenyon, G.L.,(1996). Biochemistry. 35. 5662-69

13. Kraulis P.J. (1991) J.Appl. Crystallogr. 24, 946-950 .

14. Merrit, E.A. & Murphy, M.E.P. (1991) Acta Crystallogr. D50, 869- 873 .

15. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-4680

16. Noren, C. J., et al, Science, 244, 182-188 (1989)

17. Segel, I. H., Enzyme Kinetics, J. Wiley & Sons, (1975)

18. Blundel, T. L. and N. L. Johnson, Protein Crystallography, Academic Press (1976)

19. Lattman, E., "Use of the Rotation and Translation Functions", in Methods in Enzymology, 115, 55-77 (1985)

20. M. G. Rossmann, ed., "The Molecular Replacement Method", Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York, (1972)

21. Travis, J., Science, 262, 1374 (1993)

22. Meng, E. C. et al., J. Comp. Chem., 13, pp. 505-524 (1992)

23. Methods in Enzymology, vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985) 24. Goodford, P. J., "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem., 28, 849-857 (1985))

25. Miranker, A. and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method." Proteins:

Structure. Function and Genetics, 11, 29-34 (1991)).

26. Goodsell, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure. Function, and Genetics, 8, 195-202 (1990).

27. Kuntz, I. D. et al., "A Geometric Approach to Macromolecule- Ligand Interactions", J. Mol. Biol, 161, 69-288 (1982).

28. Bartlett, P. A. et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules". In "Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, 182-196 (1989)).

29. Martin, Y. C, "3D Database Searching in Drug Design", J. Med. Chem., 35, 2145-2154 (1992)).

30. Bohm, H.-J., "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, 61-78 (1992).

31. Nishibata, Y. and A. Itai, Tetrahedron, 47,. 8985 (1991).

32. Cohen, N. C. et al., "Molecular Modeling Software and Methods for Medicinal Chemistry", J. Med. Chem., 33, 883-894 (1990). 33. Navia, M. A. and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, 202-210 (1992).

34. Wellner, D., Analyt. Chem., 43, 597 (1971).

35. Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).

36. Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154

37. Houbenwyl, 1987, Methods of Organic Chemistry, ed. E. Wansch Vol. 15 1 and II, Thieme, Struttgart.

38. Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA 1985).

39. Q1A expression TM, A Handbook for High-Level Expression and Purification of 6xHis-Tagged Proteins, March 1999, QUIAGEN

DETAILED FIGURE LEGENDS

Figure 1. Alignment of RmlC amino acid sequences indicating the location of conserved amino acids. Proposed substrate binding and catalytic residues are colored red. Alignment analysis was generated using ClustalW at the European Bioinformatics Institute server.

Genbank

Label Genus Species Accession Number

MT M. thermoautotrophicum AAB86256

Pyrh Pyrococcus horikoshii BAA29502 Neim Neisseria meningitides AAF40545

Yers Yersinia enterocoUtica. S35299

Salt Salmonella typhimurium P26394

Kleb Klebsiella pneumoniae AAC70776

Esch Escherichia coli. P37745 Myco Mycobacterium tuberculosis AAB66649

Syne Synechocystis sp BAA18590

Xant Xanthomonas campestris D49906

Neig Neisseria gonorrhoeae. P37763

Shig Shigella flexneri P37780 Acti Actinobacillus actinomycetemcomitans BAA28134

Serr Serratia marcescens AAC00178

Burc Burkholderia pseudomallei AAD05456

Bact Bacteroides fragilis AAD40710

Sale Salmonella choleraesuis. S23343

Figure 2. (A) Ribbon diagram of an RmlC subunit with a ball-and-stick model of complexed dTDP. The jelly-roll structural motif is shown by the green and red β-strands. The secondary structure elements are labeled as depicted in the text. This figure was prepared using Molscript (13) and Raster3D (14). (B) Stereoview of the C_a trace of a subunit of RmlC. The numbers refer to the amino acid residues. Figure 3. Overview of the dimeric structure of RmlC. Ribbon diagram of the RmlC dimer with a ball-and-stick model of complexed dTDP. Each subunit is colored differently. (A) Two-fold axis of symmetry in the plane. (B) Rotation of (A) by 90° in the plane.

Figure 4. (A) SigmaA weighted 2Fo-Fc electron density map of the final apo model after refinement at 1.5 A. (B) SigmaA weighted 2Fo-Fc electron density map of the final dTDP-complexed model after refinement at 1.75 A. Both maps have been contoured at the lσ level.

Figure 5. A Detailed view of the active site of RmlC. (A) Residues involved in binding dTDP and the location of the His64-Aspl72 catalytic dyad are shown. Residues are color-coded based on whether they originate from subunit A (yellow) or B (blue) and the catalytic triad is colored green. (B) A schematic two-dimensional structure of the active site of RmlC is shown. Residues and water molecules interacting with complexed dTDP are shown.

Claims

WE CLAIM:

1. A method for identifing an inhibitor for an RmlC enzyme, comprising the steps of:

(a) using the crystal structure of said enzyme with bound substrate analogue as shown in Figure 3 to design or select a potential inhibitor;

(b) obtaining said potential inhibitor;

(c) determining the ability of said potential inhibitor to inhibit said enzyme by contacting said potential inhibitor with said enzyme in the presence of a substrate

2. The method of claim 1 wherein the RmlC enzyme is a homodimer comprising two subunits.

3. The method for identifying an inhibitor for an RmlC enzyme as claimed in claim 2 wherein the crystal structure of the active site of the enzyme is used to design or select the potential inhibitor.

4. The method of claim 3 wherein the active site of the RmlC enzyme is formed by amino acid residues from β-strands 3 and 4 from one subunit of the RmlC enzyme with β-strands 5,6,11 & 12 from the other subunit.

5. The method of claim 4 wherein the RmlC enzyme is

Methanobacterium thermoautotrophicum RmlC.

6. The method of claim 5 wherein the amino residues which form the active site of RmlC enzyme comprises: Gln49, Asp84, Aspl44, Asp 172, Glu31, Lysl71, Glu52, Arg26, Arg61, His64, Hisl20, Cysl35, Ser53, Ser55, Serl69, Asn51, Trpl75, Phe29, Phel22, Tyrl33, Tyrl39.

7. The method of claim 6 wherein the dTDP binding site of the RmlC enzyme is used to design or select the potential inhibitor.

8. The method of claim 7 wherein the dTDP binding site comprises the amino acid residues Phe 29, Tyr 133, Tyr 139, Lys 171, Glu 31, Gin 49, Arg 61 and Arg 26

9. The method of claim 6, wherein the substrate binding site of the RmlC enzyme is used to design or select the potential inhibitor.

10. The method of claim 9 wherein the substrate binding site comprises the amino acid residues His 64, His 120, Asp 172, Asp 84 and Lys 73.

11. The method of claim 10 wherein the substrate binding site comprises the amino acid residues His 64, His 120 and Lys 73.

12. The method of claim 1 wherein amino acid residues 140 to 144 of RmlC enzyme from Methanobacterium thermoautorophicum are used to design or select the potential inhibitor.

13. The method of claim 1 wherein the amino acid residues of RmlC which are conserved among the bacterium selected from the group consisting of: Methanobacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, Aeitinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseria gonorrhoeae Yersinia enterocoUtica, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Klebisiella pneumoniae, Mycobacterium tuberculosis, Serratia marcescens, Burkholderia pseudomallei, Bacteroides fragilis and Salmonella choleraesuis.

14. The method of claim 1 wherein the amino acid residues V⁵⁹XRGLHZQ⁶⁰ _' where X is a hydrophobic residue and Z is an aromatic residue, of Methanobacterium thermoautotrophicum RmlC are used to design or select the inhibitor.

15. A method of determining the crystal structure of the RmlC of a bacterium comprising:comparing the X-ray diffraction data from the RmlC of the bacterium with that of the known crystal structure of Methanobacterium thermoautotorphicum RmlC with and/or without bound substrate analogue.

16. The method of claim 15 wherein the bacterium is selected from the group consisting of: Methanobacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, Aeitinobacillus actinomycetemcomitans, Neisseria meningitides, Neisseria gonorrhoeae Yersinia enterocoUtica, Shigella flexneri, Salmonella typhimurium, Escherichia coli, Klebisiella pneumoniae, Mycobacterium tuberculosis, Serratia marcescens, Burkholderia pseudomallei, Bacteroides fragilis and Salmonella choleraesuis.

17. An inhibitor of RmlC which binds to the active site of RmlC.

18. An inhibitor of RmlC enzyme which binds to the dTDP binding site of RmlC.

19. An inhibitor of RmlC which binds to the substrate binding site of RmlC.

20. An inhibitor of RmlC enzyme which binds to amino residues 140 to 144 of Methanobacterium thermoautotrophicum RmlC or homologues therof.

21. An inhibitor of RmlC enzyme which binds to amino residues V⁵⁹XRGLHZQ⁶⁶ _' where X is a hydrophobic residue and Z is an aromatic residue RmlC.

22. An inhibitor which comprises the 3-dimensional active site of

RmlC Methanobacterium thermoutrophicum and can bind dTDP and subtrate, but which lacks epimerase activity.

23. An inhibitor which comprises the 3-dimensional dTDP and substrate binding site of RmlC and can bind dTDP and substrate, but which lacks epimerase activity.

24. An inhibitor which comprises the 3-dimensional substrate binding site of RmlC Methanobacterium thermoautrophicum and can bind substrate, but which lacks epimerase activity.

25 An inhibitor identified according to the method of claims 1 to 14.

26. An antibacterial agent comprising any one of the inhibitor of claims 17-24.

27. A method of treating a bacterial infection caused by bacteria which express RmlC by administering an effective amount of any one of the inhibitors of claims 17-24 to the infected or potentially infected site.

28. A method of preventing a bacterial infection caused by bacteria which express RmlC by administering an effective amount of any one of the inhibitors of claims 17-24 to the infected or potentially infected site.

29. A method of treating a Gram negative bacterial infection caused by bacteria having L-rhamnose in the O-antigen, by administering an effective amount of any one of the inhibitors of claims 17-24 to a patient in need thereof.

30. The method of claim 28 or 29 wherein the bacterial infection is caused by the bacteria selected from the group consisting of : Mehtanobacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, A, actinomycetemcomitans, neisseria meningitides, Yersinia enterocoUtica, Shigella Glexneri, Slmonella Typhimurium, Escherichia coli, Klebisiella pneumoniae, Mucobacterium tuberculosis.

31. A use of any one of the inhibitors of claims 17-24 in the preparation of a medicament for the treatment of a bacterial infection caused by bacteria which express RmlC.

32. A use of any one of the inhibitors of claims 17-24 in the preparation of a medicament for the prevention of a bacterial infection caused by bacteria which express RmlC.

33. A use of any one of the inhibitors of claims 17-24 in the preparation of a medicament for the treatment of a Gram negative bacterial infection caused by bacteria having L-rhamnose in the O- antigen.

34. The use of claim 31 or 32 wherein the bacterial infection is caused by the bacteria selected from the group consisting of : Mehtanobacterium thermoautotrophicum, Synechocystis sp., Xanthomonas campestris, Pyrococcus hoikoshii, A, actinomycetemcomitans, neisseria meningitides, Yersinia enterocoUtica, Shigella Glexneri, Slmonella Typhimurium, Escherichia coli, Klebisiella pneumoniae, Mucobacterium tuberculosis.

35. A pharmaceutical composition comprising an inhibitor of any one of claims 17-24 and a pharmaceutically acceptable carrier.

36. An inhibitor designed or identified using anyone of the methods claimed in claims 1-16.