Crystal structure of yqeJ and uses thereof Number:7,155,346 from the United States Patent and Trademark Office (PTO) owispatent This invention is directed to the crystal structure of yqeJ, and to the use of this structure in rational drug design methods to identify agents that may interact with active sites of yqeJ. Such agents may be useful as antibacterial agents.<H structure, thereof, Crystal, structu, 7155346.html, Patent, pto, 7155346

Title: Crystal structure of yqeJ and uses thereof

Abstract: This invention is directed to the crystal structure of yqeJ, and to the use of this structure in rational drug design methods to identify agents that may interact with active sites of yqeJ. Such agents may be useful as antibacterial agents.

Patent Number: 7,155,346 Issued on 12/26/2006 to Olland, et al.

Inventors: Olland; Andrea M. (Medford, MA), Stahl; Mark L. (Lexington, MA), Sullivan; Francis X. (Boulder, CO), Underwood; Kathryn W. (Quincy, MA), Chopra; Rajiv (Andover, MA)
Assignee: Wyeth (Madison, NJ)
Millennium Pharmaceuticals, Inc. (Cambridge, MA)

Appl. No.: 10/209,041

Filed: July 31, 2002

Current U.S. Class: 702/19 ; 435/15; 702/27

Current International Class: G01N 33/48 (20060101); C12Q 1/48 (20060101)

Field of Search: 702/19,27 435/15,4,32

References Cited [Referenced By]

U.S. Patent Documents


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Primary Examiner: Nashed; Nashaat T.
Attorney, Agent or Firm: Fish & Richardson P.C.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/313,153, filed Aug. 17, 2001 and U.S. Provisional Application No. 60/340,613, filed Dec. 7, 2001.

Claims

What is claimed is:

1. A method for identifying an agent that interacts with Bacillus subtilis nicotinic acid mononucleotide adenylyl transferase yqeJ, comprising: obtaining a crystallized yqeJ of SEQ ID NO:26 comprising four molecules of yqeJ of SEQ ID NO:26, wherein the crystallized yqeJ belongs to space group P2.sub.1 with unit cell parameters a=43.98 .ANG., b=126.10 .ANG., c=70.58 .ANG., and .beta.=92.73 .ANG.; determining the three dimensional structure of yqeJ; employing said three-dimensional structure to design or select an agent that interacts with yqeJ; and obtaining the agent.

2. The method of claim 1, wherein the three dimensional structure comprises the relative structural coordinates of yqeJ of SEQ ID NO:26 as represented in FIG. 9, .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 of not more than 1.5 .ANG..

3. The method of claim 2, wherein the .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 is not more than 1.0 .ANG..

4. The method of claim 2, wherein the .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 is not more than 0.5 .ANG..

5. The method of claim 1, further comprising: contacting the identified agent with yqeJ in order to determine the effect the agent has on yqeJ activity.

6. A method for identifying an agent that interacts with Bacillus subtilis nicotinic acid mononucleotide adenylyl transferase yqeJ, comprising: obtaining a crystallized complex comprising six molecules of yqeJ of SEQ ID NO:26 and six molecules of nicotinic acid adenine dinucleotide (NaAD), wherein the crystallized complex belongs to space group P2.sub.12.sub.12.sub.1 with unit cell parameters a=78.39 .ANG., b=108.90 .ANG., c=178.09 .ANG.; determining the three dimensional structure of the crystallized complex; employing said three-dimensional structure to design or select an agent that interacts with yqeJ; and obtaining the agent.

7. The method of claim 6, wherein the three dimensional structure comprises the relative structural coordinates of yqeJ of SEQ ID NO:26 and NaAD as represented in FIG. 10, .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 of not more than 1.5 .ANG..

8. The method of claim 7, wherein the .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 is not more than 1.0 .ANG..

9. The method of claim 7, wherein the .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 is not more than 0.5 .ANG..

10. The method of claim 6, further comprising: contacting the identified agent with yqeJ in order to determine the effect the agent has on yqeJ activity.

11. A method for identifying a potential inhibitor of Bacillus subtilis nicotinic acid mononucleotide adenylyl transferase yqeJ, comprising: obtaining (i) a crystallized yqeJ of SEQ ID NO:26 comprising four molecules of yqeJ of SEQ ID NO:26, wherein the crystallized yqeJ belongs to space group P2.sub.1 with unit cell parameters a=43.98 .ANG., b=126.10 .ANG., c=70.58 .ANG., and .beta.=92.73 .ANG., or (ii) a crystallized complex comprising six molecules of yqeJ of SEQ ID NO:26 and six molecules of NaAD, wherein the crystallized complex belongs to space group P2.sub.12.sub.12.sub.1 with unit cell parameters a=78.39 .ANG., b=108.90 .ANG., c=178.09 .ANG.; determining the three dimensional structure of (i) the crystallized yqeJ or (ii) the crystallized complex of yqeJ and NaAD; selecting or designing a candidate inhibitor by performing computer fitting analysis of the candidate inhibitor with the three dimensional structure; and obtaining the candidate inhibitor.

12. The method of claim 11, wherein the three dimensional structure of (i) the crystallized yqeJ or (ii) the crystallized complex of yqeJ and NaAD comprises the relative structural coordinates of amino acid residues Ile6, Phe7, Gly8, Gly9, Thr10, Phe11, Asp12, Pro13, Pro14, His15 Asn16, Gly17, His18, Leu19, Leu20, Met21, Ala22, Val25, Phe36, Met37, Pro38, Asn39, Glu40, Ile41, Pro42, Pro43, His44, Lys45, Tyr50, Thr51, Arg56, Glu76, Pro82, Ser83, Tyr84, Thr85, Phe86, Asp87, Thr88, Phe103, Ile104, Ile105, Gly106, Ala107, Asp108, Met109, Ile110, Tyr112, Leu113, Pro114, Lys115, Trp116, Tyr117, Lys118, Leu119, Leu122, Phe128, Ile129, Gly130, Val131, Lys132, Arg133, Pro134, Phe136, Val149, Pro150, Glu151, Phe152, Glu153, Val154, Ser155, Ser156, Thr157, Met158, Ile159, Arg160 and Tyr187 of yqeJ of SEQ ID NO:26 according to FIG. 9 or 10, .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 of not more than 1.5 .ANG..

13. The method of claim 12, wherein the .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 is not more than 1.0 .ANG..

14. The method of claim 12, wherein the .+-. a root mean square deviation from the backbone atoms of yqeJ of SEQ ID NO:26 is not more than 0.5 .ANG..

15. The method of claim 11, which further comprises: introducing the candidate inhibitor in a reaction with (i) yqeJ, (ii) nicotinic acid mononucleotide (NAMN) or nicotinamide mononucleotide (NMN), and (iii) ATP; and determining the effect the candidate inhibitor has on the reaction.

16. The method of claim 11, which further comprises: introducing the candidate inhibitor in a reaction with (i) yqeJ, (ii) nicotinic acid adenine dinucleotide (NAAD) or nicotinomide adenine dinucleotide (NAD), and (iii) pyrophosphate; and determining the effect the candidate inhibitor has on the reaction.

Description

FIELD OF THE INVENTION

The present invention relates to the identification of Bacillus subtilis yqeJ as a NaMN AT, the determination of the crystal structures of yqeJ alone and bound to NaAD, and the use of the structures for designing new antimicrobial agents.

BACKGROUND OF THE INVENTION

Nicotinamide adenine dinucleotide (NAD) is an essential molecule in cells. In addition to its role in oxidation-reduction reactions, in which NAD(H) and its phosphorylated form, NADP(H), act as hydride donors and acceptors, NAD is also important for other cellular processes, such as the activity of NAD-dependent DNA ligases, mono and poly ADP-ribosylation of proteins, and production of the intracellular calcium-mobilizing molecules cADPR and NaADP (1), (2).

NAD is synthesized via a multi-step de novo pathway or via a pyridine salvage pathway. The enzyme nicotinic acid mononucleotide adenylyl transferase (NaMN AT, EC 2.7.7.18) sits at the convergence of these two pathways. NaMN AT catalyzes the conversion of ATP and nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) (FIG. 1), which is directly processed to NAD by NAD synthetase. The nadD gene, encoding NaMN AT, was the first enzyme demonstrated to be essential of NAD biosynthesis by both the de novo and salvage pathways (3). A number of enzymes demonstrating in vitro adenylyltransferase activity for NaMN and NMN have been identified in eukarya, archaea and bacteria (4), (5), (6), (7), (8), (9), (10), (11). Along with sequence homology, the specificity of these enzymes for NMN versus NaMN provides a useful method for classifying new genes within this family.

While there is sequence conservation between the eubacterial nadD genes (FIG. 2), sequence alignment of nadD NaMN ATs to the eukaryotic enzymes or archeal enzymes is difficult outside of the region surrounding the (H/T)XGH (SEQ ID NO:18) nucleotidyl transferase consensus sequence. Adenylyltransferases encoded by the nadD gene prefer the nicotinic acid containing NaMN over NMN as a substrate by a factor that ranges from 6 1 to 2000 1 (12), (13), (4). Eubacteria also contain enzymes that demonstrate higher specificity for the nicotinamide containing NMN. This group the products of the nadR gene, which in addition to its regulatory role in NAD biosynthesis, also contains NMN AT activity (14). The eukaryotic and archeal NMN AT (EC 2.7.7.1), such as those from human (15), Methanococcus jannaschii (16) and Methanobacterium thermoautotrophicum (17), either demonstrate higher specificity for NMN as a substrate, as compared to NaMN, or show little preference for either substrate (4).

Primary sequence studies indicate that NaMN AT belongs to the nucleotidyltransferase .alpha./.beta. phosphodiesterases superfamily of enzymes that contain the (H/T)XGH (SEQ ID NO:18) signature motif. Members of this family share the same basic catalytic mechanism, involving direct nucleophilic attack upon an .alpha.-phosphate followed by the release of pyrophosphate, while the enzyme provides stabilization of the transition state prior to the formation of a new phosphodiester bond. The recent structure determination of NMN ATs, from Methanococcus jannaschii and Methanobacterium thermoautotrophicum, has allowed this sequence and functional homology to be extended to the structural conservation of residues involved in substrate binding and catalysis (16), (17).

Genes that have been identified to be essential for bacterial survival are currently being evaluated for their potential as targets for anti-microbial chemotherapy. Understanding the biochemical, physical and structural properties of these essential enzymes and placing them in a larger biological context are the first steps in exploring this potential. The present invention is based on the identification of an unassigned reading frame in B. subtilis (yqeJ) as a NaMN AT. The recombinant enzyme was expressed in E. coli and shown to prefer NaMN as a substrate to NMN, allowing the assignment of it as the nadD gene of B. subtilis. It differs from the NMN ATs from Metanococcus jannaschii and Methanobacterium thermoautotrophicum both in its substrate specificity and oligomeric state. It is homodimeric as opposed to a homo-hexamer (16), (17). The three dimensional structure of NaMN AT from B. subtilis has been determined to 2.2 .ANG. and 3.2 .ANG. with the NaAD bound. This has allowed the identification of key residues in substrate binding and catalysis. These structures will provide invaluable information in the ongoing development of anti-microbial agents targeting NAD biosynthesis.

SUMMARY OF THE INVENTION

The present invention provides a crystallized yqeJ having four molecules of yqeJ (molecules A, B, C and D as set forth in FIG. 9) in the asymmetric unit. The present invention also provides a crystallized complex of yqeJ and NaAD, that includes six molecules of yqeJ (molecules A, B, C, D, E and F as set forth in FIG. 10) in the asymmetric unit.

The present invention also provides a three dimensional model of yqeJ as derived by x-ray diffraction data of the yqeJ crystal. Specifically, the three dimensional model of yqeJ is defined by the relative structural coordinates for molecules A, B, C and/or D of yqeJ (SEQ ID NO:26) according to FIG. 9, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG.. The three dimensional model of yqeJ is useful for a number of applications, including, but not limited to, the visualization, identification and characterization of various active sites of yqeJ, including the substrate binding sites of yqeJ. The active site structures may then be used to design various agents which interact with yqeJ, as well as yqeJ complexed with a substrate or related molecules.

The present invention also provides a three dimensional model of yqeJ as derived by x-ray diffraction data of the yqeJ/NaAD crystal. Specifically, the three dimensional model of yqeJ is defined by the reactive structural coordinates for molecules A, B, C, D, E and/or F of yqeJ (SEQ ID NO:26) according to FIG. 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG.. The three dimensional model of yqeJ is useful for a number of applications, including, but not limited to, the visualization, identification and characterization of various active sites of yqeJ, including the substrate binding sites of yqeJ. The active site structures may then be used to design various agents which interact with yqeJ, as well as yqeJ complexed with NaAD, other substrates or related molecules.

The present invention is also directed to an active site of yqeJ, and preferably the substrate binding site of yqeJ, which is most preferably NaAD. More preferably, the active site comprises the relative structural coordinates of amino acid residues Ile6, Phe7, Gly8, Gly9, Thr10, Phe11, Asp12, Pro13, Pro14, His15, Asn16, Gly17, His18, Leu19, Leu20, Met21, Ala22, Val25, Phe36, Met37, Pro38, Asn39, Glu40, Ile41, Pro42, Pro43, His44, Lys45, Tyr50, Thr51, Arg56, Glu76, Pro82, Ser83, Tyr84, Thr85, Phe86, Asp87, Thr88, Phe103, Ile104, Ile105, Gly106, Ala107, Asp108, Met109, Ile110, Tyr112, Leu113, Pro114, Lys115, Trp116, Tyr117, Lys118, Leu119, Leu122, Phe128, Ile129, Gly130, Val131, Lys132, Arg133, Pro134, Phe136, Val149, Pro150, Glu151, Phe152, Glu153, Val154, Ser155, Ser156, Thr157, Met158, Ile159, Arg160 and Tyr187 for molecules A, B, C or D of yqeJ (SEQ ID NO:26) according to FIG. 9 or 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG.. The active site may correspond to the configuration of yqeJ in its state of association with its substrate (e.g, NaAD) or in its unbound state.

In addition, the present invention provides a method for identifying an agent that interacts with yqeJ, comprising the steps of: (a) generating a three dimensional model of molecules A, B, C and/or D of yqeJ (SEQ ID NO:26) using the relative structural coordinates according to FIG. 9, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG.; and (b) employing said three-dimensional model to design or select an agent that interacts with yqeJ.

The present invention also provides a method for identifying an agent that interacts with yqeJ, comprising the steps of: (a) generating a three dimensional model of molecules A, B, C, D, E and/or F of yqeJ (SEQ ID NO:26) using the relative structural coordinates according to FIG. 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG.; and (b) employing said three-dimensional model to design or select an agent that interacts with yqeJ.

Still further, the present invention provides a method for identifying a potential inhibitor of yqeJ, comprising the steps of: (a) generating a three dimensional model of said molecule comprising a substrate binding site using the relative structural coordinates of amino acid residues Ile6, Phe7, Gly8, Gly9, Thr10, Phe11, Asp12, Pro13, Pro14, His15, Asn16, Gly17, His18, Leu19, Leu20, Met21, Ala22, Val25, Phe36, Met37, Pro38, Asn39, Glu40, Ile41, Pro42, Pro43, His44, Lys45, Tyr50, Thr51, Arg56, Glu76, Pro82, Ser83, Tyr84, Thr85, Phe86, Asp87, Thr88, Phe103, Ile104, Ile105, Gly106, Ala107, Asp108, Met109, Ile110, Tyr112, Leu113, Pro114, Lys115, Trp116, Tyr117, Lys118, Leu119, Leu122, Phe128, Ile129, Gly130, Val131, Lys132, Arg133, Pro134, Phe136, Val149, Pro150, Glu151, Phe152, Glu153, Val154, Ser155, Ser156, Thr157, Met158, Ile159, Arg160 and Tyr187 for molecules A, B, C or D of yqeJ (SEQ ID NO:26) according to FIG. 9 or 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG.; and (b) selecting or designing a candidate agent by performing computer fitting analysis of the candidate inhibitor with the three dimensional model generated in step (a).

Finally, the present intention provides agents or inhibitors identified using the foregoing methods. Small molecules or other agents which inhibit or otherwise interfere with substrate binding to yqeJ may be useful as antimicrobial agents.

Additional objects of the present invention will be apparent from the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the reaction catalyzed by yqeJ NaMN AT. NaMN AT catalyzes the reversible addition of Nicotinic Acid Mononucleotide (NaMN) or Nicotinamide Mononucleotide (NMN) to ATP yielding pyrophosphate and Nicotinic Acid Adenine Dinucleotide (NaAD) or Nicotinamide Adenine Dinucleotide (NAD), respectively.

FIG. 2 provides a sequence alignment of Bacillus subtilis yqeJ (SEQ ID NO:1) with known eubacterial nadD genes: Escherichia coli (SEQ ID NO:2), Bacillus halodurans (SEQ ID NO:3), Lactococcus lactis (SEQ ID NO:4), Mycobacterium leprae (SEQ ID NO:5), Streptomyces coelicolor (SEQ ID NO:6), Mycobacterium tuberculosis (SEQ ID NO:7), Treponema pallidum (SEQ ID NO:8), Borrelia burgdorferi (SEQ ID NO:9), Neisseria meningitides (SEQ ID NO:10), and Pseudomonas aeruginosa (SEQ ID NO:11). Residues that are highlighted in bold are identical, residues that are boxed (beginning at H) designate the nucleotidyl transferase consensus sequence, while the residues in the box (beginning at S) designate the SXXXXR/K motif (SEQ ID NO:20). The secondary structure elements of B. subtilis NaMN AT (SEQ ID NO:1) are overlaid on the sequence with the arrows and cylinders representing the .beta. strands and .alpha. helices, respectively.

FIGS. 3A, 3B, 3C and 3D provides the results of HPLC analysis of yqeJ NaMN AT adenylyl transferase activity. 0.36 mg/ml yqeJ NaMN AT was incubated with 1 mM ATP and 1 mM NaMN (3A) or 1 mM NMN (3B) for 30 minutes. The reverse reaction with 0.18 mg/ml yqeJ NaMN AT was assayed with 1 mM sodium pyrophosphate and either 0.5 mM NaAD (3C) or 0.5 mM NAD (3D) for 30 minutes. Under these conditions NaMN eluted at 1.16 min, NMN at 1.29 min, ATP at 2.12 min, NaAD at 5.37, and NAD at 5.47 min.

FIGS. 4A 4F provide representative data of B. subtilis NaMN AT activity. Plots show rate vs. substrate concentration: (4A) NaMN, (4B) NAAD, (4C) NMN, (4D) NAD, (4E) ATP and (4F) pyrophosphate. Lines represent fit to rate equation indicated in Methods. Forward reaction, graphs 4A, 4C and 4E, uses purine ribonucleoside phosphorylase coupled assay system. Back reaction, graphs 4B, 4D and 4F, uses the hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme assay system.

FIG. 5 provides a topology diagram of the secondary structure elements of yqeJ NaMN AT. Grey arrows and black cylinders represent .beta.-sheets and .alpha.-helices, respectively. The white cylinder represents an .alpha.-helical turn and the white arrow an isolated .beta. bridge that is part of the dimer interface.

FIGS. 6A 6F show a comparison of enzymes of the nucleotidyl-transferase .alpha./.beta. phosphodiesterases. 6A) B. subtilis NaMN AT, 6B) E. coli phosphopantetheine adenylyltransferase, 6C) M. jannaschii NMN AT, 6D) M. thermoautotrophicum NMN AT, 6E (B. subtilis glycerol-3-phosphate cytidyltransferase, and 6F) E. coli glytaminyl tRNA synthetase. Strands of the central parallel .beta.-sheet, the HXGH motif (SEQ ID NO:19) and the SxxxxR/K motif (SEQ ID NO:20) are also shown.

FIGS. 7A and 7B represent ribbon diagrams of NaMN AT. A) Dimer of dimers observed in the apo crystal structure, the two `functional` dimers are shown. B) The `functional` dimer (lower structure from FIG. 7A) is shown bound to NaAD.

FIG. 8A is an .alpha.-carbon trace of NaMN AT monomer bound to NaAD. Inset: electron density from a 3f.sup.o 2f.sub.c composite omit map is contoured at 1 .sigma.. FIG. 8B is a schematic of selected active site interactions of NaAD with NaMN AT. MC: indicates main chain interaction, SC: indicates side chain interaction.

FIG. 9 provides the atomic structural coordinates for yqeJ NaMN AT (SEQ ID NO:26) as derived by X-ray diffraction of a yqeJ crystal. "Atom type" refers to the atom whose coordinates are being measured. "Residue" refers to the type of residue of which each measured atom is a part--i.e., amino acid, cofactor, ligand or solvent. The "x, y and z" coordinates indicate the Cartesian coordinates of each measured atom's location in the unit cell (.ANG.). "Occ" indicates the occupancy factor. "B" indicates the "B-value", which is a measure of how mobile the atom is in the atomic structure (.ANG..sup.2). Under "Residue type", "A," "B," "C" and "D" refer to each molecule of yqeJ, and "S" refers to water molecules.

FIG. 10 provides the atomic structural coordinates for yqeJ NaMN AT (SEQ ID NO:26) and NaAD as derived by X-ray diffraction of the crystal complex of yqeJ NaMN AT (SEQ ID NO:26) with NaAD. "Atom type" refers to the atom whose coordinates are being measured. "Residue" refers to the type of residue of which each measured atom is a part--i.e., amino acid, cofactor, ligand or solvent. The "x, y and z" coordinates indicate the Cartesian coordinates of each measured atom's location in the unit cell (.ANG.). "Occ" indicates the occupancy factor. "B" indicates the "B-value", which is a measure of how mobile the atom is in the atomic structure (.ANG..sup.2). Under "Residue type", "A," "B," "C," "D," "E," and "F" refer to each molecule of yqeJ, and "H," "J," "K," "L," "M" and "N" refer to NaAD molecules.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms and phrases shall have the meanings set forth below:

Unless otherwise noted, yqeJ is NaMNATase and catalyzes the reversible addition of ATP to NAMN or NMN to yield pyrophosphate and NAAD or NAD, respectively. Preferably, yqeJ comprises the amino acid sequences for Bacillus subtilis depicted in FIG. 2 (SEQ ID NO:1), FIG. 9 (SEQ ID NO:26) or FIG. 10 (SEQ ID NO:26), including conservative substitutions. "YqeJ activity" refers to the ability of yqeJ to catalyze the aforementioned reaction.

A "substrate" is a compound or molecule involved in a reaction in which yqeJ acts as a catalyst, and binds to yqeJ. Preferably, the reaction is set forth in FIG. 1, and the substrate is NAMN, NMN and ATP, as well as the product of the reaction, namely, NaAD and NAD.

Unless otherwise indicated, "protein" or "molecule" shall include a protein, protein domain, polypeptide or peptide.

"Structural coordinates" are the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a molecule or molecular complex. Structural coordinates may be obtained using x-ray crystallography techniques or NMR techniques, or may be derived using molecular replacement analysis or homology modeling. Various software programs allow for the graphical representation of a set of structural coordinates to obtain a three dimensional representation of a molecule or molecular complex. The structural coordinates of the present invention may be modified from the original sets provided in FIGS. 9 and 10 by mathematical manipulation, such as by inversion or integer additions or subtractions. As such, it is recognized that the structural coordinates of the present invention are relative, and are in no way specifically limited by the actual x, y, z coordinates of FIGS. 9 and 10.

An "agent" shall include a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), molecule, compound or drug.

"Root mean square deviation" is the square root of the arithmetic mean of the squares of the deviations from the mean, and is a way of expressing deviation or variation from the structural coordinates described herein. The present invention includes all embodiments comprising conservative substitutions of the noted amino acid residues resulting in same structural coordinates within the stated root mean square deviation. It will be obvious to the skilled practitioner that the numbering of the amino acid residues of yqeJ may be different than that set forth herein, and may contain certain conservative amino acid substitutions that yield the same three dimensional structures as those defined by FIGS. 9 and 10 herein. Corresponding amino acids and conservative substitutions in other isoforms or analogues are easily identified by visual inspection of the relevant amino acid sequences or by using commercially available homology software programs (e.g., MODELLAR, MSI, San Diego, Calif.).

"Conservative substitutions" are those amino acid substitutions which are functionally equivalent to the substituted amino acid residue, either by way of having similar polarity, steric arrangement, or by belonging to the same class as the substituted residue (e.g., hydrophobic, acidic or basic), and includes substitutions having an inconsequential effect on the three dimensional structure of yqeJ with respect to the use of said structures for the identification and design of agents which interact with yqeJ and a substrate, as well as other proteins, peptides, molecules or molecular complexes comprising a substrate binding site, for molecular replacement analyses and/or for homology modeling.

An "active site" refers to a region of a molecule or molecular complex that, as a result of its shape and charge potential, favorably interacts or associates with another agent (including, without limitation, a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound or drug) via various covalent and/or non-covalent binding forces. As such, an active site of the present invention may include, for example, the actual site of substrate binding with yqeJ, as well as accessory binding sites adjacent or proximal to the actual site of substrate binding that nonetheless may affect yqeJ activity upon interaction or association with a particular agent, either by direct interference with the actual site of substrate binding or by indirectly affecting the steric conformation or charge potential of the yqeJ and thereby preventing or reducing binding of substrate to yqeJ at the actual site of substrate binding. As used herein, an "active site" also includes analog residues of yqeJ, which exhibit observable NMR perturbations in the presence of a binding ligand, such as the substrate. While such residues exhibiting observable NMR perturbations may not necessarily be in direct contact with or immediately proximate to ligand binding residues, they may be critical yqeJ residues for rational drug design protocols.

The present invention first provides a crystallized yqeJ that includes four molecules of yqeJ, namely, molecules A, B, C and D, in the asymmetric unit. In a particular embodiment, the amino acid sequence of each molecule of yqeJ (SEQ ID NO:1) is set forth in FIG. 2, and includes conservative substitutions. The crystal of the present invention effectively diffracts X-rays for the determination of the structural coordinates of yqeJ, and is characterized as having space group P2.sub.1, unit cell parameters of a=43.98 .ANG., b=126.10 .ANG., c=70.58 .ANG. and .beta.=92.73.degree.. Each molecule of yqeJ is further characterized as having the secondary structure in which .beta. and .alpha. strands are configured in trace order as 62 1, .alpha.A, .beta.2, .alpha.B, .beta.3, .alpha.C, .beta.4, .alpha.D, .beta.5, .beta.6, .alpha.E and .alpha.F. More preferably, the .beta. and .alpha. strands correspond to amino acid residues of each molecule of yqeJ (SEQ ID NO:1) as follows: .beta.1 (3 9), .alpha.A (16 28), .beta.2 (33 38), .alpha.B (53 64), .beta.3 (70 72), .alpha.C (85 95), .beta.4 (100 106), .alpha.D (117 125), .beta.5 (127 132), .beta.6 (145 148), .alpha.E (156 165) and .alpha.F (175 183).

The present invention also provides a crystallized complex of yqeJ and NaAD. The crystallized complex is preferably characterized as having space group P2.sub.12.sub.12.sub.1 with unit cell parameters a=78.39 .ANG., b=108.90 .ANG., c=178.09 .ANG., .alpha.=.beta.=.gamma.=90.00.degree. and contains six molecules of yqeJ for each molecule of NaAD in the asymmetric unit. Here again, it is preferred that the amino acid sequence of each molecule of yqeJ (SEQ ID NO:1) is set forth in FIG. 2, and includes conservative substitutions.

Using the crystals of the present invention, X-ray diffraction data can be collected by a variety of means in order to obtain the atomic coordinates of the molecules in the crystals. With the aid of specifically designed computer software, such crystallographic data can be used to generate a three dimensional structure. Various methods used to generate and refine a three dimensional structure of a molecular structure are well known to those skilled in the art, and include, without limitation, multiwavelength anomalous dispersion (MAD), multiple isomorphous replacement, reciprocal space solvent flattening, molecular replacement, and single isomorphous replacement with anomalous scattering (SIRAS).

Accordingly, the present invention also provides a three dimensional model of yqeJ as derived by x-ray diffraction data of the yqeJ crystal. The three dimensional model of yqeJ is preferably defined by the structural coordinates shown in FIG. 9 for molecules A, B, C and/or D, .+-. a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most preferably not more than 0.5 .ANG.. The three dimensional model may include all four molecules of yqeJ, each molecule alone, as well as combinations of the molecules (e.g., molecules A/B, A/C, A/D, B/C and B/D). The three dimensional model of yqeJ is useful for a number of applications, including, but not limited to, the visualization, identification and characterization of various active sites of yqeJ, including the substrate binding sites. The active site structures may then be used to design agents with interact with yqeJ, as well as yqeJ complexed with a substrate or related molecules.

The present invention also provides a three dimensional model of yqeJ as derived by x-ray diffraction data of the yqeJ/NaAD crystal complex. The three dimensional model of yqeJ is preferably defined by the structural coordinates shown in FIG. 10 for molecules A, B, C, D, E and/or F, .+-. a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most preferably not more than 0.5 .ANG.. The three dimensional model may include all six molecules of yqeJ, each molecule alone, as well as combinations of the molecules (e.g., molecules A/B, A/C, A/D, B/C and B/D, etc). However, when the model is used to define the active binding site, it is preferred that the three dimensional model is defined by the structural coordinates of the individual molecules A, B, C or D in FIG. 10 since the coordinates for these molecules are more resolved than molecules E and F. The three dimensional model of yqeJ is useful for a number of applications, including, but not limited to, the visualization, identification and characterization of various active sites of yqeJ, including the substrate binding sites. The active site structures may then be used to design agents with interact with yqeJ, as well as yqeJ complexed with NaAD, other substrates or related molecules.

The present invention is also directed to an active site of yqeJ, and preferably the substrate binding site of yqeJ. More preferably, the active site comprises the relative structural coordinates of amino acid residues Ile6, Phe7, Gly8, Gly9, Thr10, Phe11, Asp12, Pro13, Pro14, His15, Asn16, Gly17, His18, Leu19, Leu20, Met21, Ala22, Val25, Phe36, Met37, Pro38, Asn39, Glu40, Ile41, Pro42, Pro43, His44, Lys45, Tyr50, Thr51, Arg56, Glu76, Pro82, Ser83, Tyr84, Thr85, Phe86, Asp87, Thr88, Phe103, Ile104, Ile105, Gly106, Ala107, Asp108, Met109, Ile110, Tyr112, Leu113, Pro114, Lys115, Trp116, Tyr117, Lys118, Leu119, Leu122, Phe128, Ile129, Gly130, Val131, Lys132, Arg133, Pro134, Phe136, Val149, Pro150, Glu151, Phe152, Glu153, Val154, Ser155, Ser156, Thr157, Met158, Ile159, Arg160 and Tyr187 for molecules A, B, C or D of yqeJ (SEQ ID NO:26) according to FIG. 9 or 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most preferably not more than 0.5 .ANG.. The active site may correspond to the configuration of yqeJ in its state of association with a substrate such as NaAD, or in its unbound state.

Another aspect of the present invention is directed to a method for identifying an agent that interacts with yqeJ, comprising the steps of: (a) generating a three dimensional model of molecules A, B, C or D of yqeJ (SEQ ID NO:26) using the relative structural coordinates according to FIG. 9, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most preferably not more than 0.5 .ANG.; and (b) employing said three-dimensional model to design or select an agent that interacts with yqeJ.

In another embodiment, the present invention is directed to a method for identifying an agent that interacts with yqeJ, comprising the steps of: (a) generating a three dimensional model of molecules A, B, C, D, E and/or F of yqeJ (SEQ ID NO:26) using the relative structural coordinates according to FIG. 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most preferably not more than 0.5 .ANG.; and (b) employing said three-dimensional model to design or select an agent that interacts with yqeJ.

In the foregoing methods, the agent may be identified using computer fitting analyses utilizing various computer software programs that evaluate the "fit" between the putative active site and the identified agent, by (a) generating a three dimensional model of the putative active site of a molecule or molecular complex using homology modeling or the atomic structural coordinates of the active site, and (b) determining the degree of association between the putative active site and the identified agent. Three dimensional models of the putative active site may be generated using any one of a number of methods known in the art, and include, but are not limited to, homology modeling as well as computer analysis of raw date generated using crystallographic or spectroscopy data. Computer programs used to generate such three dimensional models and/or perform the necessary fitting analyses include, but are not limited to: GRID (Oxford University, Oxford, UK), MCSS (Molecular Simulations, San Diego, Calif.), AUTODOCK (Scripps Research Institute, La Jolla, Calif.), DOCK (University of California, San Francisco, Calif.), Flo99 (Thistlesoft, Morris Township, N.J.), Ludi (Molecular Simulations, San Diego, Calif.), QUANTA (Molecular Simulations, San Diego, Calif.), Insight (Molecular Simulations, San Diego, Calif.), SYBYL (TRIPOS, Inc., St. Louis. Mo.) and LEAPFROG (TRIPOS, Inc., St. Louis, Mo.). The structural coordinates also may be used to visualize the three-dimensional structure of yqeJ using MOLSCRIPT (Kraulis, P J, J. Appl. Crystallogr. 24: 946 950 (1991)) and RASTER3D (Bacon, D. J. and Anderson, W. F., J. Mol. Graph. 6: 219 220 (1998)), for example.

The effect of such an agent identified by computer fitting analyses on yqeJ activity may be further evaluated by contacting the identified agent with yqeJ and measuring the effect of the agent on yqeJ activity. Depending upon the action of the agent on the active site of yqeJ, the agent may act either as an inhibitor or activator of yqeJ activity. For example, enzymatic assays may be performed and the results analyzed to determine whether the agent is an inhibitor of yqeJ and a substrate (i.e., the agent may reduce or prevent binding affinity between yqeJ and a substrate) or an activator of yqeJ and a substrate (i.e., the agent may increase binding affinity between yqeJ and a substrate). However, in the preferred embodiment, the agent is an inhibitor of yqeJ activity. In this regard, the candidate inhibitor can be tested by obtaining the inhibitor and introducing the inhibitor in a reaction with yqeJ, NAMN (or MMN) and ATP, and determining the effect the candidate inhibitor has on the reaction. Alternatively, the candidate inhibitor can be tested by introducing the candidate inhibitor in a reaction with yqeJ, NAAD (or NAD) and pyrophosphate, and determining the effect the candidate inhibitor has on the reaction. The specific adenylyl trasferase assays that can be used are described in Example 1 below. Once a candidate inhibitor has been tested and shown to inhibit the reaction, further tests may be performed to evaluate the potential therapeutic efficacy of the agent as an antimicrobial agent.

Still further, the present invention provides a method for identifying a potential inhibitor of yqeJ, comprising the steps of: (a) generating a three dimensional model of said molecule comprising a substrate binding site using the relative structural coordinates of amino acid residues Ile6, Phe7, Gly8, Gly9, Thr10, Phe11, Asp12, Pro13, Pro14, His15, Asn16, Gly17, His18, Leu19, Leu20, Met21, Ala22, Val25, Phe36, Met37, Pro38, Asn39, Glu40, Ile41, Pro42, Pro43, His44, Lys45, Tyr50, Thr51, Arg56, Glu76, Pro82, Ser83, Tyr84, Thr85, Phe86, Asp87, Thr88, Phe103, Ile104, Ile105, Gly106, Ala107, Asp108, Met109, Ile110, Tyr112, Leu113, Pro114, Lys115, Trp116, Tyr117, Lys118, Leu119, Leu122, Phe128, Ile129, Gly130, Val131, Lys132, Arg133, Pro134, Phe136, Val149, Pro150, Glu151, Phe152, Glu153, Val154, Ser155, Ser156, Thr157, Met158, Ile159, Arg160 and Tyr187 for molecules A, B, C or D of yqeJ (SEQ ID NO:26) according to FIG. 9 or 10, .+-. a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 .ANG., preferably not more than 1.0 .ANG., and most preferably not more than 0.5 .ANG.; and (b) selecting or designing a candidate agent by performing computer fitting analysis of the candidate inhibitor with the three dimensional model generated in step (a). Here again, once the candidate inhibitor is obtained or synthesized, the candidate inhibitor may be analyzed in various assays as discussed above.

Various molecular analysis and rational drug design techniques are further disclosed in U.S. Pat. Nos. 5,834,228, 5,939,528 and 5,865,116, as well as in PCT Application No. PCT/US98/16879, published WO 99/09148, the contents of which are hereby incorporated by reference.

The present invention is also directed to the agents or inhibitors identified using the foregoing methods. Such agents or inhibitors may be a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, or drug. Small molecules or other agents which inhibit or otherwise interfere with yqeJ and a substrate may be useful in the treatment of diseases or conditions associated with bacterial infection.

The present invention may be better understood by reference to the following non-limiting Example. The following Example is presented in order to more fully illustrate the preferred embodiments of the invention, and should in no way be construed as limiting the scope of the present invention.

EXAMPLE 1

1. Methods and Methods

Cloning and expression of B. subtilis NaMN AT. The B. subtilis yqeJ gene was PCR cloned into a modified version of pET16b to yield pML208. This E. coli expression vector has the yqeJ coding sequence downstream of the T7 RNA polymerase promoter. The expressed protein contains the peptide MGHHHHHHHHHHSSGHIEGRHMPGGS (SEQ ID NO:12) fused to Lys-2 of the native sequence. This provides a purification tag and contains the cleavage site for Factor Xa, between Arg-20 and His-21 of the peptide, resulting in the cleaved protein having an extra six amino acids on its amino termini. To produce selenomethionine labeled yqeJ, the protein was expressed in BL21(DE3) Escherichia coli at 25.degree. C. Cultures were grown in shake flasks in LeMaster's media and induced at log phase with 0.5 mM IPTG. Cultures were harvested 4 hours post induction. Unlabelled yqeJ was also expressed in BL21DE3 E. coli, but at 37.degree. C. High density expression was carried out in a Biostat C-10 bioreactor (B. Braun Biotech). The culture was induced with 1.0 mM IPTG (final) at 4.6 OD.sub.600. Cells were harvested 4 hours post induction at 9.0 OD.sub.600.

Purification and cleavage of B. subtilis NaMN AT. The purification, unless otherwise stated was performed at 4.degree. C. Bacteria were resuspended in buffer (50 mM Hepes pH 7.5, 500 mM NaCl) and lysed by passage through a Microfluidics microfluidizer. The lysate was collected and centrifuged at 20,000.times.g for 30 min. The supernatant, containing 40% of the expressed yqeJ NaMN AT (the remainder being insoluble), was applied to a Poros PI column (Applied Biosystems) that was coupled to a Ni-NTA column (Qiagen). The Ni-NTA column was washed with 50 mM imidazole and the protein was eluted with a 50 800 mM imidazole gradient. 10 mM EDTA was added to the fraction containing yqeJ NaMN AT, for 6 hours, followed by dialysis against 50 mM Tris pH 8.0, 50 mM NaCl, 2 mM CaCl.sub.2. The His-tag was removed from yqeJ NaMN AT by a six hour digestion with Factor Xa (New England Biolabs) at room temperature. The reaction was applied to a Poros HQ 50 column and the bound protein was eluted with a 0 1 M NaCl gradient. The peak fraction containing yqeJ NaMN AT was diluted and applied to a Poros S column. The flow through, containing yqeJ NaMN AT, was applied to TSK-Gel G3000 SW column (TosoHaas), equilibrated with 50 mM Hepes pH 7.5, 50 mM NaCl. Protein purity was >95%.

Adenylyl Trasferase Assays

Discontinuous HPLC assay. The discontinuous HPLC assay is based upon the assays published by Mehl et al. (12) and Balducci et al. (18). Reaction conditions were 20 mM Hepes, pH 7.4, 10 mM MgCl.sub.2, and 0.36 or 0.18 mg/ml yqeJ protein incubated at 37.degree. C. For the forward reaction, the incubations contained 1 mM ATP and 1 mM NaMN or NMN. For the reverse reaction, the incubations contained or 1 mM sodium pyrophosphate and 0.5 mM NaAD or NAD. The reactants and products were separated by chromatography on a 3.9.times.150 mm C18 column (Novapack 5 .mu.m, Waters Inc). Buffer A 100 mM potassium phosphate pH 7.5. Buffer B 100 mM potassium phosphate pH 7.5 in 20% MeOH. The elution conditions were: 0 to 3 min in 100% A, 3.0 to 3.1 min to 100% B, 3.1 to 7 min 100% B. The absorbance of reactants and products was detected at 254 nm. Under these conditions NAMN eluted at 1.16 min., NMN at 1.29 min., ATP at 2.12 min., NaAD at 5.37 min. and NAD at 5.47 min.

Continuous assay. A continuous assay to monitor the reaction in the forward direction was based upon the EnzChek pyrophosphate assay from Molecular Probes (Eugene Oreg.) In this assay, inorganic pyrophosphate produced in the forward reaction of yqeJ is cleaved by inorganic pyrophosphatase to phosphate, which is used by the second coupling enzyme, purine nucleoside phosphorylase (PNP), to convert the chromogenic substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) to ribose-1-phosphate and 2-amino-6-mercapto-7methylpurine (E.sub.360=11,000 M.sup.-1cm.sup.-1). The reaction conditions were 20 mM Hepes pH 7.5, 10 mM MgCl.sub.2, 0.2 mM MESG, 1 U PNP, 0.01 U inorganic pyrophosphatase, and 0.03 .mu.g/mL yqeJ. The reaction volume was 125 .mu.L and was carried out in a 96 well plate at room temperature using spectamax384 plus plate reader recording continuously at 360 nm (Molecular Devices, Sunnyvale Calif.). For the K.sub.M determinations, NaMN was varied from 25 to 500 .mu.M with ATP held at 2 mM and ATP was varied from 50 to 1000 .mu.M with NaMN held constant at 1 mM. All kinetic constants were determined from non-linear fits of the experimental data using the enzyme kinetic module of Sigmaplot 7.0 (SPSS, Inc. San Rafael Calif.). The reaction conditions were the same for the K.sub.M determinations for NMN except the MgCl.sub.2 concentration was increased to 50 mM and the yqeJ concentration was increased to 75 .mu.g/mL due to the lower activity against this substrate. Substrate inhibition was seen in assays using NMN as a substrate, with the double reciprocal plots curving sharply upwards above 5 mM. The data was fit to a model of substrate inhibition using the equation: v=Vmax/(1+K.sub.M/S+S/Ki). The Ki determined for NMN was 17+/-3 mM. NMN was varied from 0.5 to 10 mM with ATP held constant at 0.6 mM. The kinetic constants of yqeJ using NMN and ATP substrates also was determined with a second coupled assay system, one coupling NAD production to its reduction by alcohol dehyrogenase. This assay was described by Balducci et al (Balducci et al, 1995a) and has been used to characterize other NMN ATase. Using this assay, very similar values of K.sub.M and Vmax for NMN were obtained as was determined with the PNP coupled assay described above. The alcohol dehyrogenase coupled assay was not suitable to assays NaMN as a substrate, presumably because NaAD is not a good substrate for alcohol dehydrogenase.

The back reaction for yqeJ NaMN AT was monitored using the coupled enzyme assay of hexokinase and glucose-6-phosphate dehydrogenase (from yeast). YqeJ NaMN AT converts pryrophosphate and NaAD (or NAD) to NaMN (or NMN) and ATP. The ATP is then used by hexokinase to phosphorylate glucose to give glucose-6-phosphate and ADP. Glucose-6-phosphate is oxidized to 6-phospho-glucono-.delta.-lactone by glucose-6-phosphate dehydrogenase and NADP is reduced to NADPH. The assay is followed by the absorbance of NADPH at 340 nm. Glucose-6-phosphate dehydrogenase from baker's yeast was used because this enzyme prefers NADP to NAD as a co-substrate. Assay conditions were: 20 mM HEPES pH 7.5, 50 mM MgCl.sub.2, 1 mM NaPPi, 10 mM KCl, 5 U hexokinase, 5 U glucose-6-phosphate dehydrogenase, 1 mM glucose, 0.5 mM NADP+. YqeJ concentration is 0.0015 mg/ml for NaAD determination and 0.0075 mg/ml for NAD determination. For K.sub.M determinations, NaAD was varied from 5 to 100 .mu.M holding PPi at 1 mM, NAD was varied from 1 to 10 mM holding PPi at 1 mM and PPi was varied from 0.1 to 2 mM holding NaAD at 500 .mu.M. At high concentration of NaPPi a precipitate was observed. This limited the concentrations of PPi that could be used in the assays.

Crystallization. Crystals were grown by hanging drop vapor diffusion at 18.degree. C. in drops containing 1.5 .mu.l of protein stock solution (14 mg/ml protein, 50 mM HEPES pH 7.5, 50 mM NaCl) mixed with 1.5 .mu.l of well solution (8% PEG 3350, 100 mM MgCl.sub.2) and equilibrated against 1 ml of well solution. Block shaped crystals grew in 3 weeks, measuring approximately 50 .mu.m across. NaAD-NaMN At complex co-crystals were grown at 18.degree. C. in drops containing 1.0 .mu.l of protein stock solution (14 mg/ml protein, 2 mM NaAD, 50 mM HEPES pH 7.5, 50 mM NaCl) mixed with 1.0 .mu.l of well solution (20% PEG 3350, 100 mM MgAcetate) and 0.3 .mu.l of xylitol (30% w/v). Plate-like crystals grew in 1 3 weeks to approximately 200 .mu.m.times.50 .mu.m .times.20 .mu.m.

Data collection and processing. Crystals of the apo-form belong to the space group P2.sub.1 with unit cell parameters a=43.98 .ANG., b=126.10 .ANG., c=70.58 .ANG. and .beta.92.73.degree. (.alpha. and .gamma.=90.degree.) and contain four molecules of NaMN AT in the asymmetric unit, implying a solvent content of 58.5%. To harvest crystals, an equal volume of a solution of 35% PEG 3350, 100 mM MgCl.sub.2 was added to drops, and after equilibration for several minutes crystals were swiped through another drop of this solution and cooled rapidly in liquid nitrogen. Data collection statistics are shown in Table II. MAD data were recorded at the 5.0.2 beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory using a Quantum-4 detector. Data was collected at two energies chosen based on the measured absorption at the selinium K edge: 12661 eV (.lamda.=0.97920 .ANG.) and 12959 eV (.lamda.=0.9567 .ANG.) corresponding to maximum f'' and a remote energy, respectively. Intensities were integrated and scaled using the programs Denzo and Scalepack (19).

Crystals of the NaAD bound form belong to the space group P2.sub.12.sub.12.sub.1 with unit cell parameters a=78.39 .ANG. b=108.90 .ANG. c=178.09 .ANG., .alpha.=.beta.=.gamma.=90.00.degree. and contain six molecules of yqeJ in the asymmetric unit, implying a solvent content of 55.6%. To harvest crystals, ethylene glycol was added to the drops to 20% and after mixing, the crystals were cooled rapidly in liquid nitrogen. Data collection statistics are shown in Table II. MAD data were recorded at the 5.0.2 beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory using a Quantum-4 detector. Data was collected at two consecutive energies, based on the measured absorption at the selenium K edge: 12662 eV (.lamda.=0.97910 .ANG.) and 12863 eV (.lamda.=0.96388 .ANG.) and corresponding to maximum f'' and a remote energy. Intensities were integrated and scaled using the programs Denzo and Scalepack (19).

Phasing, model building and refinement. The apo structure was determined by the multiple wavelength anomalous dispersion (MAD) method. Initially 22 selenium sites were found with the program SOLVE (20), and phasing with these sites in CNS (21) revealed two additional sites by means of a difference fourier map. Phases were calculated from these 24 sites in CNS and SHARP (22) and improved by solvent modification with Solomon (22). ARP/WARP (23) was used to partially trace the .alpha.-carbon backbone. The complete model, with the exception of residues 42 52 in all molecules and residues 118 126 in molecules A and C, was built into the experimental map. After several iterative cycles of refinement using CNS and model improvement, water molecules were placed automatically in CNS. A simulated annealing composite-omit map was calculated to check the final model. Final R.sub.work and R.sub.free values of 22.21% and 25.44% were obtained.

The NaAD-NaMN AT complex structure was determined using a combination of MAD phases and molecular replacement using the apo enzyme structure. Initially 21 selenium sites found using the program ShakeNBake (24), and phasing with these sites in MLPhare (25)revealed 13 additional sites by means of a difference fourier map. Phases were calculated from these 34 sites and improved with solvent flattening and fourfold averaging with DM (25). This experimental map was used to build four of the six molecules in the asymmetric unit, employing strict NCS restraints. At this point, the refined structure of the apo-enzyme become available. The complex model was edited such that fragments of the high-resolution apo model were substituted wherever the apo model was in good agreement with electron density in the experimental map. MAD phases were then combined with model phases to generate improved maps, and two more molecules were identified. Density for the first four molecules found is significantly stronger than for the final two, however density for the bound NaAD is good in all 6 molecules. The final R values after releasing NCS restraints and rebuilding are R.sub.free=0.2798 and R.sub.work=0.2787.

Size exclusion chromotagraphy. 90 .mu.g of B. subtilis NaMN AT was run on a Pharmacia superose 12 H/R 10/30 column in Tris buffered Saline pH 7.5 at a flow rate of 0.5 ml/min. The retention time of B. subtilis NaMN AT was 27.3 min. To determine the apparent molecular weight of B. subtilis NaMN AT, the standards Thyroglobin 670,000, Gamma globulin 158,000, Ovalbumin 44,000, Myoglobin 17,000 and Vitamin B-12 1,350 (Biorad) were also run on the column under the same conditions.

Analytical Ultracentrifugation. Protein was pre-equilibrated by dialysis at 4.sub.CC in 20 mM HEPES and 10 mM MgCl.sub.2, pH 7.2. The partial specific volume of B. subtilis NaMN AT was calculated based on the amino acid composition, and the density of the solvent was calculated from the chemical composition of the buffer using the computer program SEDNTERP and adjusted for temperature. Sedimentation velocity experiments were performed on a Beckman XL1/XLA Analytical Ultracentrifuge operating at a rotor speed of 30,000 rpm using 400 .mu.l samples loaded into two-channel carbon-Epon centerpieces in an An-60 Ti titanium rotor preequilibrated to temperature at least 1 hour prior to each experiment. The sedimentation coefficients and molecular weights were obtained by fitting the data to the program SVEDBERG (26). Sedimentation equilibrium experiments were performed at 4.degree. C. and 20.degree. C. using a rotor speed of 18,000 rpm. Samples (400 .mu.l) were loaded into two-channel cells at 3 different protein concentrations. Scans were recorded at 4.degree. C. and 20.degree. C., and signal was detected using absorbance optics (280 nm) and interference optics. Equilibrium was judged to be achieved when no deviations in a plot of the difference between successive scans taken 3 hrs apart were observed, usually within 24 hours. Although, temperature and ligand did have a minor effect upon the apparent molecular weight as determined by analytical ultracentrifugation. The variation between the velocity and equilibrium values at the different protein concentrations can be attributed to hydrodynamic effects.

Equations. The molecular weight of the protein in the presence and absence of ligand was obtained from sedimentation equilibrium experiments using the following equation: 1) C.sub.r=C.sub.o exp [m(1-.nu..rho.).omega..sup.2(r.sup.2-r.sub.o.sup.2)/2RT]+base Where C.sub.r is absorbance at radius r; C.sub.0 is absorbance at reference radius r.sub.0; M is the molar mass of the macromolecule; .nu. is the partial specific volume of the macromolecule (mL/g); .rho. is the density of the solvent; .omega. is the angular velocity of the rotor; R is gas constant; T is temperature; and base is baseline offset. The molecular weight of yqeJ NaMN AT was obtained from sedimentation velocity experiments using equation 2: 2) M=(sRT)/(1-.nu..rho.)D Where s is the sedimentation coefficient and D is the diffusion coefficeint obtained by fitting the data to the program SVEDBERG (26). 2. Results

yqeJ encodes NaMN AT nadD. The S. pneumonia genome was sequenced and searched for essential genes that may be suitable targets for the development of anti-microbial agents. Those genes identified were then tested in B. subtilis and E. coli. An unassigned open reading frame, yqeJ that was essential in all three organisms was identified in B. subtilis. Comparisons of its amino acid sequence to genbank using the program Blast (27) revealed it has homology to a number of putative adenylyl transferases including the recently assigned nadD gene of E. coli (SEQ ID NO:2) (12). yqeJ (SEQ ID NO:1) contains the signature nucleotidyl transferase consensus sequence (H/T)XGH (SEQ ID NO:18). As can be seen in FIG. 2, B. subtilis yqeJ is closely related to E. coli nadD (SEQ ID NO:2)and other putative eubacterial NaMN ATs. The B. subtilis enzyme is more distantly related to E. coli nadR and other eukaryotic and archeal NMN ATs. Alignment of the B. subtilis NaMN AT (SEQ ID NO:1) to these species was difficult owing to little homology outside of the region around the H/TXGH (SEQ ID NO:18) consensus sequence. This later group of enzymes includes the NMN AT from M. jannaschii and M. thermoautotrophicum for which three-dimensional structures have recently been determined (17), (16). Thus, it appears that the sequence of the B. subtilis enzyme is more closely related to the group of eubacterial enzymes that prefer NaMN as a substrate than to the archeal or eukaryotic enzymes that show little preference among the substrates or prefer NMN over NaMN (13), (12), (4). To confirm that yqeJ was indeed a NaMN AT recombinant protein was expressed in E. coli and purified (see Methods). Adenylyl transferase assays were performed to determi