Novel fabh enzyme, compositions capable of binding to said enzyme and methods of use thereof

Abstract

A novel E. coli FabH crystalline structure is identified. Also disclosed are methods of identifying inhibitors of these enzymes and/or active sites, and inhibitors identified by these methods.

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The invention relates to the identification of a novel enzyme active site and methods enabling the design and selection of inhibitors of that active site.

BACKGROUND OF THE INVENTION

[0002] The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, the organization of the biosynthetic apparatus is very different. Vertebrates possess a type I fatty acid synthase (FAS) in which all of the enzymatic activities are encoded on one multifunctional polypeptide, the mature protein being a homodimer. The acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad-spectrum antibacterial agents (Rock, C. & Cronan, J. 1996. Biochimica et Biophysica Acta 1302, 1-16; Jackowski, S. 1992. In Emerging Targets in Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe & N. Georgopapadakou. Chapman & Hall, New York; Jackowski, S. et al. (1989). J. Biol. Chem. 264, 7624-7629.)

[0003] The first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. Prior to this, malonyl-ACP is synthesized from ACP and malonyl-CoA by FabD, malonyl CoA:ACP transacylase. In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively). The second step in the elongation cycle is ketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by enoyl-ACP reductase (FabI). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al, (1996), J.Biol.Chem. 271, 1833-1836).

[0004] Cerulenin and thiolactomycin are potent and selective inhibitors of bacterial fatty acid biosynthesis. Extensive work with these inhibitors has proved that this biosynthetic pathway is essential for bacterial viability. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics would be rendered inactive by known antibiotic resistance mechanisms. There is an unmet need for developing new classes of antibiotic compounds, such as those that target FabH.

[0005] FabH enzymes are of interest as potential targets for antibacterial agents.

[0006] There is a need in the art for novel FabH enzyme active sites and catalytic sequences to enable identification and structure-based design of inhibitors, which are useful in the treatment or prophylaxis of diseases, particularly diseases caused by bacteria which may share catalytic domains with those of the invention.

SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a novel FabH enzyme active site crystalline form.

[0008] In another aspect, the present invention provides a novel FabH composition characterized by the catalytic residues Cys112, His244 and Asn274.

[0009] In still another aspect, the present invention provides a novel FabH composition characterized by the active site of 33 amino acid residues (including the catalytic residues).

[0010] In yet another aspect, the invention provides a method for identifying inhibitors of the compositions described above which methods involve the steps of: providing the coordinates of the structure of the invention to a computerized modeling system; identifying compounds which will bind to the structure; and screening the compounds identified for FabH inhibitory bioactivity.

[0011] In a further aspect, the present invention provides an inhibitor of the catalytic activity of any composition bearing the catalytic domain described above.

[0012] Another aspect of this invention includes machine readable media encoded with data representing the coordinates of the three-dimensional structure of the FabH crystal.

[0013] Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 provides the atomic coordinates of the E. coli FabH dimer.

[0015] FIG. 2 provides the atomic coordinates of the E. coli FabH monomer in complex with acetyl-CoA.

[0016] FIG. 3 provides a projection of the ribbon diagram of the E. coli FabH dimer. The two monomers are drawn with a light or dark gray shading. The catalytic Cys112 is shown in dark ball-and-stick model.

[0017] FIG. 4 provides the ribbon diagram of the E. coli FabH monomer with the catalytic residue Cys112 is shown in dark ball-and-stick model. The N- and C-termini are labeled.

[0018] FIG. 5 provides the stereoview of the α-carbon superposition between the structures of FabH and FabF. FabH is drawn in a thin black line and FabF in a thick gray line.

[0019] FIG. 6 provides the ribbon diagram of the E. coli FabH monomer with acetylated Cys112 and the CoA molecule in black ball-and-stick model. The orientation of the view is the same as that of FIG. 4.

[0020] FIG. 7 provides the superposition of the E. coli FabH catalytic residues in comparison to those of FabF. FabH is drawn in thick gray lines and FabF in thin black lines. FabH residues are label Cys112, His244 and Asn274, which corresponds to Cys163, His303 and His340, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a novel E. coli FabH crystalline structure, a novel FabH active site, and methods of use of the crystalline form and active site to identify FabH inhibitor compounds (peptide, peptidomimetic or synthetic compositions) characterized by the ability to competitively inhibit binding to the active site of a FabH enzyme. Also provided herein is a novel FabH crystalline structure in complex with the substrate acetyl-CoA, and the identification of acetyl-CoA interacting residues in FabH.

[0022] I. The Novel FabH Crystalline Three-Dimensional Structure

[0023] The present invention provides a novel FabH crystalline structure based on the E. coli FabH. The amino acid sequences of the FabH are provided in Table 1 as SEQ ID NO:1.

TABLE 1
Met Tyr Thr Lys Ile Ile Gly Thr Gly Ser Tyr Leu Pro Glu Gln
1               5                   10                  15
Val Arg Thr Asn Ala Asp Leu Glu Lys Met Val Asp Thr Ser Asp
16              20                 25                 30
Glu Trp Ile Val Thr Arg Thr Gly Ile Arg Glu Arg His Ile Ala
31              35                 40                 45
Ala Pro Asn Glu Thr Val Ser Thr Met Gly Phe Glu Ala Ala Thr
46              50                 55                 60
Arg Ala Ile Glu Met Ala Gly Ile Glu Lys Asp Gln Ile Gly Leu
61              65                 70                 75
Ile Val Val Ala Thr Thr Ser Ala Thr His Ala Phe Pro Ser Ala
76              80                 85                 90
Ala Cys Gln Ile Gln Ser Met Leu Gly Ile Lys Gly Cys Pro Ala
91              95                100                105
Phe Asp Val Ala Ala Ala Cys Ala Gly Phe Thr Tyr Ala Leu Ser
106             110               115                120
Val Ala Asp Gln Tyr Val Lys Ser Gly Ala Val Lys Tyr Ala Leu
121             125               130                135
Val Val Gly Ser Asp Val Leu Ala Arg Thr Cys Asp Pro Thr Asp
136             140               145                150
Arg Gly Thr Ile Ile Ile Phe Gly Asp Gly Ala Gly Ala Ala Val
151             155               160                165
Leu Ala Ala Ser Glu Glu Pro Gly Ile Ile Ser Thr His Leu His
166             170               175                180
Ala Asp Gly Ser Tyr Gly Glu Leu Leu Thr Leu Pro Asn Ala Asp
181             185               190                195
Arg Val Asn Pro Glu Asn Ser Ile His Leu Thr Met Ala Gly Asn
196             200               205                210
Glu Val Phe Lys Val Ala Val Thr Glu Leu Ala His Ile Val Asp
211             215               220                225
Glu Thr Leu Ala Ala Asn Asn Len Asp Arg Ser Gln Leu Asp Trp
226             230               235                240
Leu Val Pro His Gln Ala Asn Leu Arg Ile Ile Ser Ala Thr Ala
241             245               250                255
Lys Lys Leu Gly Met Ser Met Asp Asn Val Val Val Thr Leu Asp
256             260               265                270
Arg His Gly Asn Thr Ser Ala Ala Ser Val Pro Cys Ala Leu Asp
271             275               280                285
Glu Ala Val Arg Asp Gly Arg Ile Lys Pro Gly Gln Leu Val Leu
286             290               295                300
Leu Glu Ala Phe Gly Gly Gly Phe Thr Trp Gly Ser Ala Leu Val Arg Phe
301             305               310                       317

[0024] As illustrated herein, the crystal structure is a tightly associated FabH dimer. Each monomer has two structural domains: the N-terminal domain (residues 1-170 of SEQ ID NO:1) and the C-terminal domain (residues 171-317 of SEQ ID NO:1). The two domains are similar in their overall fold: each contains a 5-stranded β-sheet sandwiched between α-helices and covered by other β-strands, α-helices and loops. The structural similarity between the two halves of the protein indicates that FabH is probably evolved from two genes of similar origin. The active site of FabH is at the center of the FabH monomer, formed at the junction of the N- and C-terminal domains. While the core architecture of the E. coli FabH bears some similarity to that of the FabF (Huang, et al, (1998), EMBO J. 17, 1183-1191), large differences exit in the atomic positions of the core β-strands, and the structures outside of the core β-strand are completely different. With amino acid sequence identity between FabH and FabF being below 20%, the large differences are well expected. Therefore, the crystalline structure of E. coli FabH is novel.

[0025] As described above, the E. coli FabH is a dimer, each monomer contains an active site. The dimer formation is essential for the FabH activity because the active site of a monomer is comprised of at least Phe87 of the other monomer in the dimer. The present invention provides both a crystalline monomer and dimer structure of E. coli FabH. Inhibitors that perturb or interact with this dimer interface are another target for the design and selection of anti-bacterial agents.

[0026] According to the present invention, the crystal structure of E. coli FabH has been resolved at 2.0 Å (crystal form 1), and its selenomethionine mutant protein in complex with acetyl-CoA has been determined at 1.9 Å (crystal form 2). The structure was determined using the methods of MAD phasing and molecular replacement, and refined to R-factors of 18.9% and 27%, respectively.

[0027] Further refinement of the atomic coordinates will change the numbers in FIG. 1-2 and Tables I-III, refinement of the crystal structure from another crystal form will result in a new set of coordinates. However, distances and angles in Tables II will remain the same within experimental errors, and relative conformation of residues in the active site will remain the same within experimental error. For example, the two independently determined monomers in our crystal form 1 and the monomer in crystal form 2 do not have identical numerical coordinates, but the structures of these three monomers have very similar structures, and the spatial relationship between amino acid residues are considered the same within experimental error. In fact, we would consider any structure that can be superimposed onto that of FabH with an rms error of less than 1.5 Å on α-carbon atoms being a close structural homologue and the same rms error but over all protein atoms being an identical structure. FIG. 1 provides the atomic coordinates of the E. coli FabH dimer, which contains 634 amino acids. FIG. 2 provides the atomic coordinates of the E. coli FabH monomer in complex with acetyl-CoA, which contains 317 amino acids. The FabH enzyme is characterized by an active site which preferably contains a binding site for the first substrate acetyl-CoA and the second substrate malonyl-ACP. The catalytic residues in FabH are Cys112, His244 and Asn274, compared to Cys163, His303 and His340 in FabF. The difference in catalytic residues is not only limited to their amino acid identity (His340 to Asn274 change), but also their relative spatial arrangement. While FabH Cys112 and Asn274 can be well superimposed onto FabF Cys163 and His340, His244 of FabH occupies a very different position from that of His303 of FabF. This indicated the catalytic mechanisms of the two enzymes are very different. The crystal structure described herein was solved in the presence and absence of acetyl-CoA. We identified that the catalytic Cys112 has been covalently aceytlated, and the product CoA is still bound to the active site. The bound CoA enabled us to identify the active site cavity, which is long and narrow and shaped nicely to bind the β-mercaptoethylamine-patotheinate arm of CoA. The structure of the acetyl-CoA complex also revealed all the key residues that are interacting with CoA and lining the active site, which is identified as a set of 33 amino acid residues listed in Table I. For example, the adenine part of CoA is sandwiched between the side chains of Arg151and Trp32. Our structures are determined in the absence of malonyl-ACP. However, the same acetyl-CoA binding cavity should bind malonyl-ACP as well because their active site binding regions are very similar and there is no apparent additional entrance to the active site. Moreover, while the FabH molecular surface in general negatively charged, a region just outside of the active site cavity is positively charge. This surface is mainly comprised of three α-helices (30-37, 209-231 and 248-258) and contains a number of positively charged amino acids (Arg36, Arg40, Lys214, His222, Arg235 Arg249, Lys256, Lys257). Since the acyl-carrier protein (ACP) is known to be very acidic or negatively charged, it is reasonable to assume this surface being the ACP binding surface.

[0028] Table I provides the the atomic coordinates of the apo E. coli FabH structure in the active site (in crystal form 1). Solvent molecules are omitted here for clarity, but can be found in FIG. 1. Residue 487 is Phe87 from the other monomer.

TABLE I
ATOM	RESIDUE	X	Y	Z	Occ	B
1	N	THR	28	−24.151	18.846	61.990	1.00	36.45
2	CA	THR	28	−23.735	19.054	60.610	1.00	36.69
3	CB	THR	28	−22.196	19.086	60.565	1.00	32.66
4	OG1	THR	28	−21.760	20.076	59.636	1.00	33.79
5	CG2	THR	28	−21.645	17.737	60.183	1.00	27.40
6	C	THR	28	−24.238	17.990	59.627	1.00	38.85
7	O	THR	28	−24.732	16.923	60.023	1.00	42.97
8	N	TRP	32	−24.091	20.068	53.681	1.00	30.06
9	CA	TRP	32	−23.725	21.413	54.092	1.00	28.93
10	CB	TRP	32	−24.277	21.708	55.486	1.00	29.27
11	CG	TRP	32	−24.036	23.126	55.939	1.00	31.13
12	CD2	TRP	32	−22.895	23.622	56.644	1.00	32.44
13	CE2	TRP	32	−23.118	25.005	56.890	1.00	35.25
14	CE3	TRP	32	−21.707	23.038	57.096	1.00	32.45
15	CD1	TRP	32	−24.880	24.197	55.779	1.00	33.86
16	NE1	TRP	32	−24.333	25.331	56.351	1.00	35.49
17	CZ2	TRP	32	−22.200	25.800	57.565	1.00	35.24
18	CZ3	TRP	32	−20.793	23.832	57.765	1.00	34.43
19	CH2	TRP	32	−21.046	25.197	57.994	1.00	36.72
20	C	TRP	32	−22.203	21.582	54.091	1.00	27.24
21	O	TRP	32	−21.675	22.617	53.674	1.00	26.75
22	N	ILE	33	−21.503	20.566	54.581	1.00	26.32
23	CA	ILE	33	−20.042	20.617	54.642	1.00	25.89
24	CB	ILE	33	−19.459	19.370	55.333	1.00	25.18
25	CG2	ILE	33	−17.925	19.444	55.366	1.00	26.64
26	CG1	ILE	33	−20.024	19.253	56.744	1.00	18.01
27	CD1	ILE	33	−19.621	18.008	57.421	1.00	19.10
28	C	ILE	33	−19.432	20.755	53.258	1.00	24.76
29	O	ILE	33	−18.630	21.650	53.022	1.00	23.20
30	N	ARG	36	−20.198	24.159	51.621	1.00	26.35
31	CA	ARG	36	−19.545	25.296	52.237	1.00	27.73
32	CB	ARG	36	−20.083	25.473	53.649	1.00	34.96
33	CG	ARG	36	−19.562	26.715	54.326	1.00	47.48
34	CD	ARG	36	−20.581	27.250	55.290	1.00	56.04
35	NE	ARG	36	−21.775	27.729	54.600	1.00	63.48
36	CZ	ARG	36	−22.490	28.780	54.996	1.00	67.12
37	NH1	ARG	36	−23.564	29.153	54.303	1.00	67.75
38	NH2	ARG	36	−22.127	29.465	56.082	1.00	68.27
39	C	ARG	36	−18.014	25.292	52.233	1.00	23.26
40	O	ARG	36	−17.386	26.346	52.208	1.00	21.26
41	N	THR	37	−17.423	24.103	52.214	1.00	20.79
42	CA	THR	37	−15.973	23.969	52.258	1.00	19.72
43	CB	THR	37	−15.549	23.164	53.509	1.00	20.01
44	OG1	THR	37	−16.014	21.812	53.384	1.00	17.59
45	CG2	THR	37	−16.157	23.752	54.765	1.00	18.21
46	C	THR	37	−15.363	23.272	51.047	1.00	20.77
47	O	THR	37	−14.234	23.571	50.657	1.00	20.98
48	N	CYS	112	−0.698	28.695	58.467	1.00	12.58
49	CA	CYS	112	−0.984	28.096	57.174	1.00	11.86
50	CB	CYS	112	−2.457	28.264	56.808	1.00	10.86
51	SG	CYS	112	−3.580	27.460	57.935	1.00	22.06
52	C	CYS	112	−0.126	28.620	56.037	1.00	10.86
53	O	CYS	112	−0.003	27.939	55.025	1.00	13.89
54	N	LEU	142	−3.033	20.066	62.705	1.00	16.58
55	CA	LEU	142	−4.063	20.954	63.207	1.00	17.95
56	CB	LEU	142	−4.281	22.159	62.287	1.00	15.72
57	CG	LEU	142	−3.100	23.125	62.126	1.00	18.13
58	CD1	LEU	142	−3.628	24.499	61.738	1.00	14.84
59	CD2	LEU	142	−2.246	23.204	63.415	1.00	12.26
60	C	LEU	142	−5.396	20.321	63.598	1.00	17.45
61	O	LEU	142	−6.111	20.883	64.417	1.00	17.68
62	N	ARG	151	−17.927	23.092	65.249	1.00	22.20
63	CA	ARG	151	−18.230	22.887	63.841	1.00	25.49
64	CB	ARG	151	−19.699	23.217	63.534	1.00	24.14
65	CG	ARG	151	−20.051	22.998	62.052	1.00	33.87
66	CD	ARG	151	−21.530	23.158	61.748	1.00	37.44
67	NE	ARG	151	−21.991	24.545	61.780	1.00	41.79
68	CZ	ARG	151	−23.272	24.897	61.737	1.00	44.63
69	NH1	ARG	151	−23.612	26.173	61.771	1.00	46.51
70	NH2	ARG	151	−24.219	23.970	61.666	1.00	47.88
71	C	ARG	151	−17.304	23.634	62.868	1.00	26.00
72	O	ARG	151	−16.686	23.018	61.992	1.00	26.64
73	N	GLY	152	−17.164	24.940	63.077	1.00	24.63
74	CA	GLY	152	−16.353	25.769	62.201	1.00	23.08
75	C	GLY	152	−14.912	25.371	61.944	1.00	22.21
76	O	GLY	152	−14.366	25.679	60.880	1.00	21.32
77	N	ILE	155	−14.484	20.649	60.878	1.00	18.82
78	CA	ILE	155	−14.866	20.149	59.564	1.00	18.77
79	CB	ILE	155	−16.223	20.733	59.071	1.00	17.77
80	CG2	ILE	155	−17.365	20.321	60.018	1.00	12.79
81	CG1	ILE	155	−16.127	22.249	58.924	1.00	15.46
82	CD1	ILE	155	−17.339	22.892	58.331	1.00	20.95
83	C	ILE	155	−13.823	20.489	58.531	1.00	18.45
84	O	ILE	155	−13.819	19.909	57.461	1.00	21.51
85	N	ILE	156	−12.958	21.450	58.819	1.00	18.70
86	CA	ILE	156	−11.985	21.825	57.812	1.00	19.10
87	CB	ILE	156	−11.999	23.375	57.499	1.00	24.79
88	CG2	ILE	156	−13.391	23.974	57.563	1.00	23.59
89	CG1	ILE	156	−11.095	24.139	58.438	1.00	24.77
90	CD1	ILE	156	−9.886	24.631	57.730	1.00	27.97
91	C	ILE	156	−10.544	21.338	57.935	1.00	18.32
92	O	ILE	156	−9.922	21.071	56.918	1.00	18.31
93	N	PHE	157	−10.005	21.200	59.142	1.00	16.26
94	CA	PHE	157	−8.611	20.780	59.280	1.00	15.33
95	CB	PHE	157	−7.984	21.371	60.551	1.00	15.71
96	CG	PHE	157	−7.868	22.858	60.523	1.00	19.05
97	CD1	PHE	157	−8.814	23.654	61.158	1.00	19.74
98	CD2	PHE	157	−6.844	23.476	59.814	1.00	15.77
99	CE1	PHE	157	−8.737	25.057	61.076	1.00	21.28
100	CE2	PHE	157	−6.761	24.855	59.727	1.00	11.63
101	CZ	PHE	157	−7.701	25.650	60.351	1.00	17.65
102	C	PHE	157	−8.278	19.286	59.190	1.00	16.07
103	O	PHE	157	−9.045	18.413	59.622	1.00	17.36
104	N	LEU	189	−7.786	34.391	64.172	1.00	19.01
105	CA	LEU	189	−7.338	33.021	63.922	1.00	19.46
106	CB	LEU	189	−6.897	32.907	62.463	1.00	23.06
107	CG	LEU	189	−6.422	31.587	61.872	1.00	23.21
108	CD1	LEU	189	−7.435	30.493	62.157	1.00	24.24
109	CD2	LEU	189	−6.253	31.811	60.355	1.00	25.52
110	C	LEU	189	−6.164	32.746	64.850	1.00	18.26
111	O	LEU	189	−5.082	33.338	64.688	1.00	15.62
112	N	LEU	205	−7.765	25.549	68.834	1.00	19.58
113	CA	LEU	205	−7.699	26.448	67.677	1.00	19.69
114	CB	LEU	205	−7.475	25.601	66.398	1.00	19.66
115	CG	LEU	205	−7.104	26.238	65.052	1.00	19.36
116	CD1	LEU	205	−6.309	25.259	64.201	1.00	18.01
117	CD2	LEU	205	−8.366	26.671	64.321	1.00	18.66
118	C	LEU	205	−8.996	27.273	67.597	1.00	17.98
119	O	LEU	205	−10.088	26.731	67.804	1.00	20.78
120	N	MET	207	−11.189	30.405	65.330	1.00	16.50
121	CA	MET	207	−11.285	31.040	64.025	1.00	18.56
122	CB	MET	207	−11.105	30.003	62.931	1.00	20.76
123	CG	MET	207	−11.293	30.550	61.542	1.00	23.66
124	SD	MET	207	−10.858	29.292	60.353	1.00	32.43
125	CE	MET	207	−12.262	28.166	60.555	1.00	31.26
126	C	MET	207	−12.599	31.742	63.776	1.00	18.83
127	O	MET	207	−13.666	31.152	63.934	1.00	19.82
128	N	GLY	209	−14.190	32.425	61.134	1.00	20.42
129	CA	GLY	209	−14.305	32.056	59.737	1.00	23.52
130	C	GLY	209	−14.623	33.114	58.701	1.00	24.44
131	O	GLY	209	−13.771	33.456	57.884	1.00	26.52
132	N	ASN	210	−15.839	33.640	58.738	1.00	23.37
133	CA	ASN	210	−16.291	34.615	57.758	1.00	25.94
134	CB	ASN	210	−17.724	35.006	58.035	1.00	24.49
135	CG	ASN	210	−18.633	33.818	58.029	1.00	25.13
136	OD1	ASN	210	−18.680	33.068	57.061	1.00	25.86
137	ND2	ASN	210	−19.325	33.603	59.130	1.00	25.81
138	C	ASN	210	−15.426	35.831	57.639	1.00	26.62
139	O	ASN	210	−15.214	36.334	56.545	1.00	27.59
140	N	VAL	212	−12.110	35.950	58.414	1.00	25.15
141	CA	VAL	212	−10.808	35.645	57.793	1.00	25.13
142	CB	VAL	212	−10.004	34.469	58.486	1.00	23.52
143	CG1	VAL	212	−10.492	34.190	59.896	1.00	20.23
144	CG2	VAL	212	−9.958	33.220	57.653	1.00	23.20
145	C	VAL	212	−10.971	35.405	56.272	1.00	23.49
146	O	VAL	212	−10.095	35.769	55.493	1.00	20.62
147	N	PHE	213	−12.115	34.859	55.853	1.00	22.05
148	CA	PHE	213	−12.371	34.627	54.431	1.00	22.19
149	CB	PHE	213	−13.718	33.954	54.244	1.00	20.95
150	CG	PHE	213	−14.116	33.771	52.794	1.00	23.47
151	CD1	PHE	213	−14.758	34.788	52.101	1.00	22.38
152	CD2	PHE	213	−13.833	32.587	52.132	1.00	21.51
153	CE1	PHE	213	−15.098	34.634	50.784	1.00	23.71
154	CE2	PHE	213	−14.173	32.423	50.813	1.00	26.06
155	CZ	PHE	213	−14.805	33.446	50.133	1.00	25.34
156	C	PHE	213	−12.307	35.935	53.645	1.00	22.07
157	O	PHE	213	−11.618	36.045	52.629	1.00	22.83
158	N	ALA	216	−8.801	37.118	53.586	1.00	18.14
159	CA	ALA	216	−7.964	36.216	52.808	1.00	19.00
160	CB	ALA	216	−8.183	34.775	53.218	1.00	17.94
161	C	ALA	216	−8.146	36.371	51.303	1.00	17.97
162	O	ALA	216	−7.166	36.285	50.563	1.00	16.52
163	N	LEU	220	−4.879	37.537	49.135	1.00	15.55
164	CA	LEU	220	−4.379	36.571	48.174	1.00	17.76
165	CB	LEU	220	−5.127	35.233	48.275	1.00	15.75
166	CG	LEU	220	−4.703	34.362	49.466	1.00	13.65
167	CD1	LEU	220	−5.621	33.177	49.608	1.00	13.89
168	CD2	LEU	220	−3.278	33.915	49.310	1.00	9.45
169	C	LEU	220	−4.491	37.186	46.769	1.00	17.28
170	O	LEU	220	−3.618	36.957	45.932	1.00	20.62
171	N	HIS	244	−3.012	27.197	48.689	1.00	18.90
172	CA	HIS	244	−3.165	26.587	49.988	1.00	17.18
173	CB	HIS	244	−2.914	27.594	51.111	1.00	17.15
174	CG	HIS	244	−3.178	27.035	52.465	1.00	17.42
175	CD2	HIS	244	−2.579	26.015	53.138	1.00	14.15
176	ND1	HIS	244	−4.285	27.385	53.212	1.00	16.36
177	CE1	HIS	244	−4.370	26.596	54.264	1.00	16.12
178	NE2	HIS	244	−3.354	25.760	54.244	1.00	19.14
179	C	HIS	244	−4.631	26.151	49.971	1.00	15.41
180	O	HIS	244	−5.503	26.936	49.591	1.00	14.10
181	N	ALA	246	−7.440	26.051	51.721	1.00	19.76
182	CA	ALA	246	−8.240	26.512	52.864	1.00	20.02
183	CB	ALA	246	−8.166	28.017	52.975	1.00	22.28
184	C	ALA	246	−9.687	26.075	52.772	1.00	23.08
185	O	ALA	246	−10.281	25.620	53.759	1.00	23.37
186	N	ASN	247	−10.280	26.349	51.615	1.00	21.20
187	CA	ASN	247	−11.645	25.983	51.311	1.00	22.48
188	CB	ASN	247	−12.653	26.733	52.190	1.00	24.84
189	CG	ASN	247	−12.700	28.195	51.888	1.00	26.54
190	OD1	ASN	247	−13.343	28.618	50.942	1.00	32.63
191	ND2	ASN	247	−12.016	28.987	52.686	1.00	31.62
192	C	ASN	247	−11.824	26.292	49.825	1.00	23.43
193	O	ASN	247	−11.076	27.097	49.249	1.00	23.61
194	N	ARG	249	−14.126	27.939	48.119	1.00	27.79
195	CA	ARG	249	−14.566	29.305	47.811	1.00	28.98
196	CB	ARG	249	−15.376	29.912	48.966	1.00	34.43
197	CG	ARG	249	−16.577	29.118	49.433	1.00	45.16
198	CD	ARG	249	−17.307	29.859	50.557	1.00	52.72
199	NE	ARG	249	−18.235	30.862	50.037	1.00	60.09
200	CZ	ARG	249	−18.607	31.976	50.675	1.00	62.73
201	NH1	ARG	249	−19.469	32.803	50.096	1.00	64.82
202	NH2	ARG	249	−18.112	32.290	51.867	1.00	60.24
203	C	ARG	249	−13.369	30.208	47.562	1.00	24.80
204	O	ARG	249	−13.358	31.007	46.629	1.00	24.09
205	N	ILE	250	−12.393	30.135	48.453	1.00	24.48
206	CA	ILE	250	−11.201	30.951	48.306	1.00	24.93
207	CB	ILE	250	−10.365	30.965	49.621	1.00	26.91
208	CG2	ILE	250	−8.880	31.128	49.350	1.00	22.69
209	CG1	ILE	250	−10.902	32.091	50.506	1.00	32.57
210	CD1	ILE	250	−10.216	32.245	51.828	1.00	38.42
211	C	ILE	250	−10.391	30.533	47.076	1.00	23.12
212	O	ILE	250	−10.024	31.380	46.265	1.00	20.24
213	N	ASN	274	−7.884	20.993	55.104	1.00	16.04
214	CA	ASN	274	−6.887	22.042	55.213	1.00	16.17
215	CB	ASN	274	−7.524	23.307	55.790	1.00	16.71
216	CG	ASN	274	−6.524	24.433	56.031	1.00	15.26
217	OD1	ASN	274	−5.290	24.259	55.970	1.00	14.69
218	ND2	ASN	274	−7.058	25.607	56.319	1.00	17.12
219	C	ASN	274	−5.800	21.538	56.144	1.00	18.93
220	O	ASN	274	−6.016	21.456	57.366	1.00	18.02
221	N	SER	276	−2.883	23.010	56.745	1.00	14.34
222	CA	SER	276	−1.996	24.086	57.152	1.00	14.51
223	CB	SER	276	−1.772	23.993	58.686	1.00	16.80
224	OG	SER	276	−1.051	25.104	59.218	1.00	17.07
225	C	SER	276	−0.675	24.141	56.352	1.00	13.90
226	O	SER	276	−0.719	24.199	55.132	1.00	15.64
227	N	ALA	303	−0.360	31.072	49.683	1.00	15.48
228	CA	ALA	303	−0.934	30.617	50.937	1.00	13.31
229	CB	ALA	303	0.045	29.692	51.624	1.00	11.10
230	C	ALA	303	−1.261	31.801	51.853	1.00	12.59
231	O	ALA	303	−0.614	32.842	51.789	1.00	11.22
232	N	PHE	304	−2.299	31.642	52.666	1.00	14.82
233	CA	PHE	304	−2.726	32.650	53.626	1.00	15.34
234	CB	PHE	304	−4.075	33.248	53.207	1.00	17.57
235	CG	PHE	304	−4.561	34.355	54.119	1.00	23.99
236	CD1	PHE	304	−5.356	34.060	55.243	1.00	22.77
237	CD2	PHE	304	−4.220	35.687	53.866	1.00	22.34
238	CE1	PHE	304	−5.794	35.064	56.089	1.00	19.22
239	CE2	PHE	304	−4.657	36.705	54.712	1.00	24.60
240	CZ	PHE	304	−5.447	36.389	55.826	1.00	19.92
241	C	PHE	304	−2.831	31.946	54.982	1.00	15.89
242	O	PHE	304	−3.176	30.768	55.041	1.00	14.69
243	N	GLY	305	−2.490	32.637	56.065	1.00	14.20
244	CA	GLY	305	−2.583	32.002	57.363	1.00	13.74
245	C	GLY	305	−2.765	32.889	58.578	1.00	13.32
246	O	GLY	305	−2.788	34.115	58.496	1.00	12.57
247	N	GLY	306	−2.856	32.235	59.727	1.00	17.80
248	CA	GLY	306	−3.033	32.929	60.990	1.00	17.87
249	C	GLY	306	−1.928	33.892	61.357	1.00	19.45
250	O	GLY	306	−0.758	33.751	60.965	1.00	19.00
251	N	PHE	487	0.118	30.521	66.721	1.00	16.05
252	CA	PHE	487	−0.254	31.597	65.800	1.00	15.49
253	CB	PHE	487	−0.539	31.168	64.330	1.00	10.60
254	CG	PHE	487	−1.559	30.100	64.167	1.00	9.77
255	CD1	PHE	487	−1.169	28.788	63.944	1.00	11.44
256	CD2	PHE	487	−2.916	30.410	64.157	1.00	12.27
257	CE1	PHE	487	−2.109	27.796	63.715	1.00	10.46
258	CE2	PHE	487	−3.878	29.416	63.925	1.00	11.90
259	CZ	PHE	487	−3.477	28.111	63.705	1.00	9.96
260	C	PHE	487	−1.381	32.376	66.460	1.00	13.45
261	O	PHE	487	−2.233	31.776	67.132	1.00	15.58

[0029] Table II provides the distances between (D) atoms of the active site residues that are within 5.0 angstroms of one another as defined by Table 1.

TABLE II
		Distance
		Between			Between
Atom 1	Atom 2	(D=)	Atom 1	Atom 2	(D=)
28CA	151CD	4.796	32N	33CA	4.198
28CB	33CD1	4.204	32N	33C	4.728
28CB	151CD	4.292	32N	33CB	4.967
28CB	33CG1	4.398	32CA	33N	2.428
28CB	151CG	4.703	32CA	33CA	3.808
28OG1	33CG1	3.472	32CA	33C	4.422
28OG1	33CD1	3.709	32CA	33CB	4.890
28OG1	151CD	3.743	32CB	33N	3.133
28OG1	32CE3	3.902	32CB	33CA	4.454
28OG1	151CG	4.159	32CG	33N	3.849
28OG1	32CZ3	4.306	32CG	33CA	4.892
28OG1	155CG2	4.418	32CD2	33N	3.941
28OG1	32CD2	4.776	32CD2	36CB	4.506
28OG1	33CB	4.930	32CD2	36CD	4.511
28OG1	151NE	4.962	32CD2	33CA	4.602
28CG2	33CD1	3.435	32CD2	36NE	4.722
28CG2	33CG1	4.093	32CE2	36CD	3.747
32N	33N	2.785	32CE2	36NE	3.804
32CE2	36CZ	4.270	32CZ3	33CG1	4.754
32CE2	36CB	4.465	32CZ3	151CZ	4.802
32CE2	36NH2	4.640	32CH2	36CD	3.427
32CE2	36CG	4.706	32CH2	151NE	3.956
32CE2	151CZ	4.851	32CH2	36CG	4.238
32CE2	36NH1	4.909	32CH2	36NE	4.297
32CE3	33N	3.532	32CH2	151CD	4.299
32CE3	33CA	3.828	32CH2	151CZ	4.365
32CE3	33CG1	4.157	32CH2	155CD1	4.378
32CE3	36CB	4.522	32CN2	36CB	4.459
32CE3	155CD1	4.542	32CH2	151NH1	4.669
32CE3	33CB	4.649	32CH2	151CG	4.722
32CE3	151CD	4.657	32CH2	36NH2	4.800
32CE3	36CD	4.719	32CH2	36CZ	4.890
32CE3	151NE	4.929	32C	33CA	2.430
32CD1	36NE	4.848	32C	33C	3.009
32NE1	36NE	3.919	32C	33O	3.730
32NE1	36CZ	4.139	32C	33CB	3.737
32NE1	36CD	4.346	32C	36N	4.094
32NE1	36NH1	4.404	32C	33CG1	4.149
32NE1	36NH2	4.693	32C	36CB	4.453
32CZ2	36CD	3.146	32C	36CA	4.929
32CZ2	36NE	3.563	32C	33CG2	4.950
32CZ2	36CZ	3.945	32O	33N	2.249
32CZ2	36NH2	3.954	32O	33CA	2.757
32CZ2	36CG	4.276	32O	33C	2.945
32CZ2	151CZ	4.401	32O	36N	2.962
32CZ2	151NE	4.403	32O	33O	3.261
32CZ2	15NH1	4.452	32O	36CB	3.270
32CZ2	36CB	4.464	32O	36CA	3.712
32CZ2	36NH1	4.873	32O	33CB	4.267
32CZ2	151NH2	4.924	32O	36CG	4.657
32CZ2	151CD	4.993	32O	37N	4.735
32CZ3	155CD1	3.624	32O	36C	4.758
32CZ3	151CD	4.106	32O	33CG1	4.844
32CZ3	36CD	4.225	33N	36N	4.835
32CZ3	151NE	4.250	33CA	37OG1	4.386
32CZ3	151CG	4.430	33CA	36N	4.658
32CZ3	36CB	4.488	33CA	36CB	4.957
32CZ3	33CA	4.545	33CA	37N	4.991
32CZ3	33N	4.616	33CA	37CG2	4.994
32CZ3	36CG	4.653	33CB	37OG1	4.651
33CG2	37OG1	3.631	36CB	37CG2	4.430
33CG2	155CB	4.276	36CB	37CA	4.592
33CG2	155CD1	4.585	36CG	37N	3.982
33CG2	155O	4.633	36CG	37CG2	4.535
33CG2	37CG2	4.695	36CG	37CA	4.970
33CG2	155CG2	4.767	36C	37CA	2.432
33CG2	37CB	4.789	36C	37CG2	3.497
33CG2	155CG1	4.874	36C	37CB	3.498
33CG1	155CG2	4.351	36C	37C	3.538
33CG1	155CB	4.696	36C	37OG1	4.176
33CG1	155CD1	4.793	36C	37O	4.442
33CD1	155CG2	4.145	36C	249CD	4.916
33CD1	155CB	4.658	36C	249CG	4.954
33C	37OG1	3.580	36O	37N	2.243
33C	36N	3.854	36O	37CA	2.766
33C	37N	4.042	36O	37CG2	3.844
33C	37CB	4.576	36O	37C	3.859
33C	36CA	4.656	36O	249CD	3.882
33C	37CG2	4.688	36O	37CB	3.898
33C	36CB	4.779	36O	249CG	4.005
33C	37CA	4.826	36O	37O	4.477
33C	36C	4.863	36O	247CB	4.749
33O	37OG1	2.646	36O	247OD1	4.807
33O	37N	2.851	36O	37OG1	4.881
33O	36N	3.274	37CA	247CB	4.321
33O	37CB	3.467	37CA	247CA	4.867
33O	37CA	3.608	37CB	156CG2	4.663
33O	37CG2	3.684	37CB	247CB	4.782
33O	36C	3.777	37CG2	155CD1	3.854
33O	36CA	3.840	37CG2	156CG2	3.941
33O	36CB	4.138	37CG2	155CG1	4.422
33O	37C	4.148	37CG2	156CB	4.991
33O	36O	4.926	37C	247CB	4.542
36N	37N	2.838	37C	247CA	4.609
36N	37CA	4.277	37C	247C	4.810
36N	37C	4.949	37O	247CA	3.598
36CA	37N	2.434	37O	247C	3.729
36CA	37CA	3.811	37O	247CB	3.853
36CA	37CG2	4.500	37O	247N	4.926
36CA	37CB	4.704	37O	247O	4.938
36CA	37C	4.796	112N	276OG	3.686
36CB	37N	3.318	112N	305CA	3.963
112N	306N	4.333	112SG	304O	4.414
112N	305C	4.677	112SG	157CE2	4.485
112N	304O	4.709	112SG	274CG	4.632
112N	276CB	4.828	112SG	276N	4.659
112N	305N	4.952	112SG	305CA	4.685
112N	276CA	4.966	112SG	276C	4.686
112CA	276OG	3.624	112SG	244ND1	4.776
112CA	276C	4.051	112SG	142CD1	4.820
112CA	304O	4.061	112C	304O	3.861
112CA	276CA	4.136	112C	305CA	4.386
112CA	305CA	4.225	112C	304C	4.415
112CA	276O	4.408	112C	276C	4.524
112CA	244NE2	4.434	112C	303CB	4.545
112CA	276CB	4.443	112C	276O	4.551
112CA	244CE1	4.710	112C	244CD2	4.605
112CA	304C	4.800	112C	305N	4.661
112CA	244CD2	4.813	112C	244NE2	4.671
112CA	305N	4.911	112C	276OG	4.831
112CB	304O	3.148	112C	244CG	4.958
112CB	244CE1	3.594	112O	244CD2	3.728
112CB	244NE2	3.694	112O	276O	3.809
112CB	305CA	3.781	112O	303CB	3.827
112CB	304C	4.127	112O	244NE2	4.073
112CB	244ND1	4.129	112O	276C	4.079
112CB	276OG	4.216	112O	244CG	4.177
112CB	276CA	4.217	112O	304O	4.251
112CB	244CD2	4.306	112O	244CE1	4.632
112CB	305N	4.436	112O	244ND1	4.683
112CB	276C	4.515	112O	276CA	4.831
112CB	244CG	4.571	112O	244CB	4.890
112CB	276CB	4.716	112O	304C	4.905
112CB	276O	4.728	112O	304N	4.955
112CB	306N	4.945	112O	303CA	4.975
112CB	305C	4.962	142CA	157CB	4.754
112CB	274OD1	4.977	142CA	205CD1	4.956
112SG	276OG	3.687	142CB	157CD2	3.797
112SG	276CA	3.809	142CB	157CG	4.058
112SG	244CE1	3.853	142CB	157CB	4.165
112SG	276CB	3.982	142CB	205CD1	4.170
112SG	244NE2	4.070	142CB	157CE2	4.469
112SG	274OD1	4.127	142CB	276CB	4.757
112SG	274ND2	4.259	142CB	157CD1	4.905
142CG	276CB	3.788	151C	155N	4.563
142CG	276OG	4.071	151C	155CD1	4.597
142CG	205CD1	4.377	151C	155CB	4.899
142CG	157CD2	4.414	151O	152N	2.258
142CG	157CE2	4.706	151O	152CA	2.779
142CD1	276CB	3.608	151O	152C	2.947
142CD1	276OG	3.655	151O	155CG1	3.212
142CD1	205CD1	3.719	151O	155CG2	3.411
142CD1	157CE2	3.740	151O	155N	3.421
142CD1	157CD2	3.885	151O	152O	3.701
142CD1	487CZ	4.116	151O	155CD1	3.721
142CD1	487CE1	4.134	151O	155CB	3.737
142CD1	157CZ	4.454	151O	155CA	4.176
142CD1	157CG	4.706	152CA	155CG1	4.815
142CD1	276CA	4.885	152CA	155CD1	4.922
142CD2	487CE1	4.604	152C	207CE	4.094
142CD2	205CD1	4.620	152C	155CG1	4.510
142CD2	276OG	4.759	152C	156CG2	4.843
142CD2	276CB	4.818	152C	155N	4.860
142C	157CB	4.133	152O	207CE	3.274
142C	157CG	4.691	152O	156CG2	3.855
142O	157CB	4.323	152O	155CG1	4.323
142O	205CD1	4.386	152O	156CG1	4.363
142O	157CG	4.706	152O	156CB	4.727
151N	152N	2.952	152O	155CD1	4.807
151N	152CA	4.351	152O	156N	4.911
151CA	152N	2.436	155N	156N	2.685
151CA	152CA	3.810	155N	156CA	4.127
151CA	152C	4.558	155N	156CG2	4.821
151CA	155CG2	4.685	155N	157N	4.835
151CB	152N	3.099	155N	156C	4.966
151CB	152CA	4.414	155CA	156N	2.427
151CG	152N	3.627	155CA	156CA	3.765
151CG	155CG2	4.303	155CA	156CG2	4.562
151CG	155CD1	4.606	155CA	156C	4.769
151CG	152CA	4.623	155CA	156CB	4.784
151CD	152N	4.899	155CA	157N	4.991
151C	152CA	2.431	155CB	156N	3.352
151C	152C	3.097	155CB	156CA	4.554
151C	152O	4.095	155CB	156CG2	4.561
151C	155CG1	4.343	155CG2	156N	4.705
151C	155CG2	4.371	155CG1	156N	3.270
155CG1	156CG2	3.509	156CG1	157CE2	4.578
155CG1	156CA	4.310	156CG1	157CB	4.670
155CG1	156CB	4.510	156CG1	207CE	4.697
155CD1	156CG2	4.165	156CG1	274ND2	4.790
155CD1	156N	4.638	156CG1	246O	4.975
155C	156CA	2.383	156CD1	274ND2	3.308
155C	156C	3.439	156CD1	274CB	3.331
155C	156CB	3.567	156CD1	157CZ	3.561
155C	156CG2	3.643	156CD1	157CE1	3.563
155C	157N	3.931	156CD1	157N	3.712
155C	156O	4.261	156CD1	157CE2	3.715
155C	156CG1	4.558	156CD1	157CD1	3.722
155O	156N	2.227	156CD1	274CG	3.772
155O	156CA	2.675	156CD1	157CD2	3.864
155O	156C	3.604	156CD1	157CG	3.875
155O	156CB	3.915	156CD1	246O	4.111
155O	156CG2	4.089	156CD1	157CA	4.343
155O	156O	4.103	156CD1	274CA	4.694
155O	157N	4.363	156CD1	157CB	4.712
156N	157N	2.981	156CD1	274N	4.913
156N	157CA	4.422	156CD1	274OD1	4.936
156CA	157N	2.466	156C	157N	1.329
156CA	157CA	3.825	156C	157CA	2.420
156CA	157C	4.700	156C	157C	3.305
156CA	157O	4.854	156C	157CB	3.660
156CA	157CB	4.870	156C	157O	3.694
156CA	157CD1	4.959	156C	274N	3.900
156CA	274N	4.980	156C	157CG	4.021
156CB	157N	3.377	156C	274CB	4.195
156CB	157CA	4.624	156C	157CD1	4.329
156CB	246O	4.688	156C	274O	4.565
156CB	274CB	4.791	156C	274CA	4.613
156CB	157CD1	4.859	156C	157CD2	4.668
156CG2	157N	4.653	156O	157N	2.229
156CG1	157N	3.213	156O	157CA	2.717
156CG1	157CD1	3.583	156O	274N	2.729
156CG1	157CE1	3.655	156O	157C	3.324
156CG1	157CG	4.050	156O	274CB	3.467
156CG1	157CZ	4.179	156O	274CA	3.614
156CG1	157CA	4.262	156O	157O	3.892
156CG1	157CD2	4.517	156O	274O	3.950
156CG1	274CB	4.523	156O	157CB	4.129
156O	274C	4.220	157CZ	205CD1	4.113
156O	157CG	4.518	157CZ	205CD2	4.153
156O	274CG	4.862	157CZ	274CG	4.640
156O	157CD2	4.863	157CZ	205CG	4.775
157N	274O	4.374	157CZ	207SD	4.820
157N	274N	4.566	157C	274O	3.627
157N	274CB	4.672	157C	274N	4.446
157CA	274O	3.295	157C	274C	4.527
157CA	274N	4.244	157O	274O	4.850
157CA	274C	4.279	189N	207CA	4.847
157CA	274CB	4.444	189CA	207CA	4.417
157CA	274CA	4.594	189CA	207N	4.864
157CB	274O	3.745	189CA	207CB	4.928
157CB	274C	4.921	189CA	487CE2	4.997
157CG	274O	3.919	189CB	306CA	4.135
157CG	205CD1	4.661	189CB	212CG1	4.600
157CG	274CB	4.767	189CB	487CE2	4.841
157CG	274CG	4.946	189CB	306N	4.926
157CD1	205CD1	4.256	189CB	487CD2	4.995
157CD1	205CD2	4.394	189CG	306CA	3.750
157CD1	205CG	4.976	189CG	487CE2	3.924
157CD2	274O	3.280	189CG	306N	4.212
157CD2	274CG	3.915	189CG	487CD2	4.347
157CD2	274CB	4.085	189CG	487CZ	4.911
157CD2	274ND2	4.099	189CG	207CG	4.991
157CD2	274OD1	4.220	189CD1	207CB	3.783
157CD2	274C	4.280	189CD1	207CG	3.907
157CD2	205CD1	4.766	189CD1	207SD	4.051
157CD2	274CA	4.819	189CD1	487CE2	4.116
157CE1	205CD2	3.643	189CD1	207CA	4.314
157CE1	205CD1	3.963	189CD1	205CD2	4.490
157CE1	205CG	4.458	189CD1	487CZ	4.872
157CE1	207CE	4.729	189CD1	207N	4.916
157CE1	207SD	4.791	189CD1	487CD2	4.942
157CE2	274ND2	3.503	189CD2	306CA	3.467
157CE2	274CG	3.728	189CD2	306N	3.480
157CE2	274OD1	4.078	189CD2	305C	4.060
157CE2	274O	4.205	189CD2	305O	4.557
157CE2	274CB	4.299	189CD2	305CA	4.739
157CE2	205CD1	4.515	189CD2	212CG2	4.797
157CE2	274C	4.976	189CD2	212CG1	4.883
157CZ	274ND2	4.083	189CD2	306C	4.903
189CD2	487CE2	4.911	209N	212N	4.914
189CD2	212CB	4.963	209CA	210N	2.421
189C	487CD2	4.060	209CA	210CA	3.796
189C	487CE2	4.144	209CA	212CG1	4.372
189C	487O	4.648	209CA	210C	4.462
189C	306CA	4.974	209CA	212N	4.662
189O	487CD2	3.681	209CA	210CB	4.826
189O	487O	4.066	209CA	212CG2	4.959
189O	487CE2	4.173	209CA	210CG	4.975
189O	487C	4.215	209C	210CA	2.434
189O	306CA	4.247	209C	210C	3.026
189O	306C	4.621	209C	210CB	3.693
189O	487CG	4.813	209C	212N	3.800
205CG	487CZ	4.299	209C	210O	3.920
205CG	487CE2	4.667	209C	210CG	4.126
205CD1	487CZ	4.050	209C	213N	4.177
205CD1	487CE2	4.824	209C	210OD1	4.376
205CD1	487CE1	4.931	209C	212CG1	4.433
205CD2	207CB	4.532	209C	213CB	4.625
205CD2	207N	4.789	209C	212CA	4.667
205C	207N	4.445	209C	210ND2	4.747
205O	207N	4.564	209C	212CG2	4.782
207CA	209N	4.326	209C	212CB	4.818
207CB	209N	4.314	209C	212C	4.948
207CB	209CA	4.966	209O	210N	2.245
207CG	209N	3.475	209O	210CA	2.777
207CG	209CA	3.821	209O	210C	2.905
207CG	212CG1	4.074	209O	213N	2.972
207CG	212CG2	4.903	209O	212N	3.043
207SD	209CA	4.461	209O	210O	3.487
207SD	209N	4.640	209O	213CB	3.674
207SD	212CG2	4.851	209O	212CA	3.685
207SD	212CG1	4.933	209O	212C	3.773
207CE	209CA	4.469	209O	212CG2	3.827
207CE	209N	4.711	209O	213CA	3.906
207C	209N	3.159	209O	212CG1	3.916
207C	209CA	4.396	209O	212CB	3.947
207O	209N	3.120	209O	210CB	4.249
207O	209CA	4.341	209O	210CG	4.878
209N	210N	3.152	209O	212O	4.958
209N	212CG1	4.281	209O	210OD1	4.993
209N	210CA	4.540	210N	212N	4.398
210N	213N	4.866	212C	216CA	4.658
210N	213CB	4.979	212C	213CG	4.966
210CA	213CB	4.405	212O	213N	2.245
210CA	212N	4.438	212O	216N	2.670
210CA	213N	4.596	212O	213CA	2.759
210C	213N	3.886	212O	213C	2.887
210C	213CB	4.239	212O	216CB	3.134
210C	213CA	4.591	212O	213O	3.255
210C	212CA	4.624	212O	216CA	3.457
210C	212C	4.679	212O	213CB	4.240
210O	213N	3.501	212O	304CE1	4.399
210O	213CB	3.633	212O	216C	4.660
210O	212N	3.644	212O	304CZ	4.701
210O	213CA	3.933	213N	216N	4.607
210O	213C	4.126	213N	216CB	4.734
210O	212C	4.352	213CA	250CD1	4.134
210O	212CA	4.631	213CA	216CB	4.363
210O	213CG	4.674	213CA	216N	4.434
210O	213CD1	4.727	213CA	250CG1	4.898
212N	213N	2.784	213CA	216CA	4.958
212N	213CA	4.205	213CB	250CD1	4.585
212N	213C	4.773	213CG	250CG1	4.288
212N	213CB	4.895	213CG	250CD1	4.298
212CA	213N	2.468	213CG	249NH2	4.361
212CA	213CA	3.845	213CD1	249NH2	4.189
212CA	213C	4.420	213CD1	250CG1	4.969
212CA	216N	4.888	213CD1	249CZ	4.976
212CA	213CB	4.891	213CD2	250CG1	3.388
212CB	213N	3.397	213CD2	250CD1	3.646
212CB	213CA	4.698	213CD2	247ND2	4.070
212CB	304CE1	4.881	213CD2	247OD1	4.172
212CG1	213N	4.408	213CD2	249NH2	4.297
212CG2	213N	3.253	213CD2	249CB	4.423
212CG2	213CA	4.264	213CD2	247CG	4.542
212CG2	304CE1	4.815	213CD2	250CB	4.579
212C	213N	1.335	213CD2	250N	4.650
212C	213CA	2.440	213CD2	249CD	4.689
212C	213C	2.994	213CD2	250CA	4.924
212C	213CB	3.710	213CE1	249NH2	3.969
212C	213O	3.755	213CE1	249CZ	4.403
212C	216N	3.855	213CE1	249NH1	4.789
212C	216CB	4.183	213CE1	250CG1	4.914
213CE1	249NE	4.963	213O	213N	3.471
213CE2	250CG1	3.302	213O	216CA	3.662
213CE2	249CB	3.341	213O	216CB	3.709
213CE2	250N	3.738	213O	216C	3.731
213CE2	213CB	3.785	213O	250CD1	4.129
213CE2	247OD1	3.897	213O	250CG1	4.545
213CE2	249C	4.015	213O	216O	4.914
213CE2	249CD	4.057	216N	304CZ	4.099
213CE2	249NH2	4.080	216N	304CE2	4.314
213CE2	250CD1	4.089	216N	304CE1	4.419
213CE2	250CA	4.157	216N	304CD2	4.807
213CE2	250CB	4.248	216N	304CD1	4.895
213CE2	249CG	4.314	216CA	304CE2	3.847
213CE2	249CA	4.346	216CA	304CD2	3.926
213CE2	249NE	4.420	216CA	304CZ	3.934
213CE2	249CZ	4.459	216CA	304CG	4.094
213CE2	247ND2	4.468	216CA	304CE1	4.099
213CE2	249O	4.492	216CA	304CD1	4.169
213CE2	247CG	4.604	216CA	250CD1	4.669
213CZ	249CB	3.765	216CA	304CB	4.908
213CZ	249NH2	3.909	216CA	220N	4.975
213CZ	249CZ	4.112	216CA	220CD1	4.997
213CZ	250CG1	4.148	216CB	250CD1	3.531
213CZ	249NE	4.295	216CB	304CD1	3.550
213CZ	249C	4.377	216CB	304CE1	3.746
213CZ	249CD	4.394	216CB	304CG	3.756
213CZ	250N	4.428	216CB	304CZ	4.110
213CZ	249O	4.508	216CB	304CD2	4.118
213CZ	249NH1	4.708	216CB	304CE2	4.288
213CZ	249CG	4.729	216CB	304CB	4.383
213CZ	250CA	4.749	216CB	250CG1	4.685
213CZ	249CA	4.754	216CB	220CD1	4.706
213C	216N	3.701	216C	220N	4.091
213C	216CB	4.305	216C	220CG	4.389
213C	216CA	4.432	216C	220CD1	4.410
213C	250CD1	4.614	216C	220CB	4.425
213C	216C	4.795	216C	250CD1	4.646
213O	216N	3.163	216C	304CD2	4.738
213O	212O	3.255	216C	304CE2	4.889
213O	213CB	3.375	216C	220CA	4.901
213O	213CG	3.382	216C	304CG	4.985
213O	213CD1	3.423	216O	220N	2.973
216O	220CB	3.240	244CG	303CB	4.261
216O	220CG	3.312	244CG	246N	4.437
216O	220CD1	3.600	244CG	303CA	4.495
216O	220CA	3.682	244CG	304O	4.536
216O	304CD2	4.466	244CG	276O	4.605
216O	220CD2	4.723	244CG	304N	4.694
216O	220C	4.729	244CG	274OD1	4.945
216O	304CG	4.812	244CD2	276O	3.276
216O	304CE2	4.867	244CD2	276C	4.179
220CG	304CB	3.954	244CD2	274OD1	4.296
220CG	304CD2	4.620	244CD2	276CA	4.491
220CG	304CG	4.655	244CD2	276N	4.705
220CG	304N	4.839	244CD2	303CB	4.764
220CG	303C	4.910	244ND1	246N	3.736
220CG	304CA	4.914	244ND1	246CB	3.939
220CG	303O	4.942	244ND1	304O	4.002
220CD1	250CG2	3.858	244ND1	246CA	4.065
220CD1	304CB	3.918	244ND1	274OD1	4.288
220CD1	304N	4.769	244ND1	274ND2	4.528
220CD1	304CG	4.781	244ND1	274CG	4.656
220CD1	304CA	4.980	244ND1	304N	4.729
220CD2	303O	3.794	244CE1	274OD1	3.036
220CD2	303C	3.874	244CE1	274ND2	3.525
220CD2	304CB	4.033	244CE1	274CG	3.527
220CD2	303N	4.091	244CE1	246N	4.024
220CD2	304N	4.170	244CE1	246CA	4.116
220CD2	303CA	4.361	244CE1	246CB	4.253
220CD2	304CA	4.531	244CE1	304O	4.409
220CD2	304CD2	4.978	244CE1	276O	4.453
220CD2	304CG	4.997	244CE1	276CA	4.503
244N	303CA	4.590	244CE1	276N	4.607
244N	303N	4.800	244CE1	274CB	4.806
244N	303CB	4.918	244CE1	276C	4.903
244CA	246N	4.644	244NE2	274OD1	2.997
244CA	303CA	4.703	244NE2	276O	3.189
244CA	303CB	4.756	244NE2	276CA	3.620
244CB	303CA	3.618	244NE2	276N	3.747
244CB	303CB	3.663	244NE2	276C	3.774
244CB	304N	4.380	244NE2	274CG	3.873
244CB	303N	4.545	244NE2	274ND2	4.248
244CB	303C	4.581	244NE2	246N	4.811
244CB	246N	4.821	244C	246N	3.311
244C	246CA	4.639	246C	274CG	4.829
244O	246N	3.012	246O	247N	2.265
244O	246CA	4.288	246O	246CA	2.400
244O	246CB	4.440	246O	247CA	2.826
246N	247N	2.858	246O	247CB	3.054
246N	247CA	4.225	246O	247ND2	3.937
246N	247O	4.519	246O	247CG	3.998
246N	274ND2	4.635	246O	274ND2	4.116
246N	274CG	4.694	246O	274CB	4.132
246N	247C	4.783	246O	247C	4.279
246N	274CB	4.908	246O	274CG	4.548
246CA	247N	2.398	246O	247O	4.812
246CA	274ND2	3.762	247CA	249N	4.491
246CA	247CA	3.780	247CB	249N	4.494
246CA	274CG	4.159	247CG	250N	3.957
246CA	274CB	4.398	247CG	249N	4.038
246CA	247CB	4.470	247CG	250CB	4.274
246CA	247ND2	4.518	247CG	249CB	4.318
246CA	247O	4.632	247CG	250CG1	4.508
246CA	247C	4.704	247CG	249CA	4.619
246CA	274OD1	4.840	247CG	249CG	4.681
246CA	247CG	4.866	247CG	250CD1	4.751
246CB	247N	3.017	247CG	250CA	4.762
246CB	247ND2	3.981	247CG	249C	4.818
246CB	274ND2	4.268	247OD1	249N	3.007
246CB	247CA	4.360	247OD1	250N	3.066
246CB	247CG	4.666	247OD1	249CB	3.116
246CB	247CB	4.733	247OD1	249CA	3.431
246CB	247O	4.816	247OD1	249CG	3.604
246CB	250CG2	4.830	247OD1	249C	3.735
246CB	250CD1	4.837	247OD1	250CB	4.015
246CB	250CB	4.978	247OD1	250CA	4.121
246CB	274CG	4.988	247OD1	249CD	4.172
246C	247CA	2.445	247OD1	250CG1	4.267
246C	247CB	3.093	247OD1	250CD1	4.870
246C	247C	3.647	247OD1	249O	4.930
246C	247ND2	3.730	247ND2	250CD1	3.820
246C	247CG	3.789	247ND2	250CG1	3.953
246C	247O	3.922	247ND2	250CB	4.004
246C	274ND2	4.440	247ND2	250N	4.402
246C	274CB	4.631	247ND2	250CA	4.869
246C	247OD1	4.815	247C	249N	3.305
247C	250N	4.120	274O	276CA	4.809
247C	249CA	4.545	303N	304CA	4.862
247C	249C	4.779	303CA	304N	2.430
247C	250CB	4.900	303CA	304CA	3.818
247C	250CA	4.940	303CA	304C	4.661
247O	249N	3.360	303CA	304O	4.679
247O	250N	3.406	303CA	304CB	4.684
247O	250CB	3.950	303CB	304N	3.222
247O	250CA	3.970	303CB	304CA	4.521
247O	250C	4.123	303CB	304O	4.818
247O	249C	4.217	303CB	304C	4.963
247O	249CA	4.373	303C	304CA	2.452
247O	250CG2	4.591	303C	304CB	3.442
249N	250N	2.817	303C	304C	3.504
249N	250CA	4.203	303C	304O	3.860
249N	250C	4.666	303C	305N	4.467
249CA	250N	2.413	303C	304CG	4.748
249CA	250CA	3.779	303O	304N	2.247
249CA	250C	4.413	303O	304CA	2.806
249CA	250CB	4.866	303O	304CB	3.762
249CB	250N	3.035	303O	304C	3.989
249CB	250CA	4.353	303O	304O	4.630
249CG	250N	4.416	303O	305N	4.674
249C	250CA	2.410	303O	304CG	4.827
249C	250C	3.035	304N	305N	3.547
249C	250CB	3.720	304N	305CA	4.719
249C	250O	3.774	304CA	305N	2.450
249C	250CG1	4.278	304CA	305CA	3.795
249C	250CG2	4.919	304CA	305C	4.958
249O	250N	2.240	304CB	305N	3.325
249O	250CA	2.733	304CG	305N	3.321
249O	250C	3.038	304CG	305CA	4.469
249O	250O	3.374	304CG	305O	4.729
249O	250CB	4.232	304CD1	305N	3.304
249O	250CG1	4.716	304CD1	305CA	4.052
274OD1	276CB	4.452	304CD1	305O	4.145
274OD1	276C	4.632	304CD1	305C	4.383
274OD1	276O	4.648	304CD2	305N	4.139
274ND2	276N	4.935	304CE1	305O	3.966
274C	276N	3.322	304CE1	305N	4.100
274C	276CA	4.688	304CE1	305C	4.483
274O	276N	3.552	304CE1	305CA	4.616
304CE2	305N	4.804
304CE2	305O	4.952
304CZ	305O	4.401
304CZ	305N	4.783
304C	305N	1.329
304C	305CA	2.395
304C	305C	3.718
304C	305O	4.130
304C	306N	4.754
304O	305N	2.239
304O	305CA	2.696
304O	305C	4.145
304O	305O	4.826
304O	306N	4.921
305N	306N	3.702
305N	306CA	4.963
305CA	306N	2.391
305CA	306CA	3.771
305CA	306O	4.400
305CA	306C	4.467
305C	306CA	2.427
305C	306C	3.071
305C	306O	3.236
305O	306N	2.248
305O	306CA	2.772
305O	306C	2.996
305O	306O	3.217
306N	487CD2	4.792
306CA	487CD2	4.048
306CA	487CG	4.502
306CA	487CB	4.525
306CA	487CE2	4.655
306C	487CB	4.265
306C	487CD2	4.576
306C	487CG	4.734
306O	487CB	4.248
306O	487CG	4.922

[0030] Table III provides the the atomic coordinates of the acetyl-CoA complex structure in the active site. Solvent molecules are omitted here for clarity, but can be found in FIG. 2. Residue 487 is Phe87 from the other monomer. Residue CAC is acetylated cysteine, and COA is the bound CoA molecule.

TABLE III
ATOM	RESIDUE	X	Y	Z	Occ	B
1	N	THR	28	32.909	0.319	26.935	1.00	14.64
2	CA	THR	28	31.524	0.759	27.053	1.00	16.73
3	CB	THR	28	31.399	2.311	26.861	1.00	18.66
4	OG1	THR	28	30.140	2.771	27.368	1.00	21.07
5	CG2	THR	28	31.523	2.702	25.394	1.00	14.87
6	C	THR	28	30.671	−0.021	26.041	1.00	15.95
7	O	THR	28	31.196	−0.755	25.190	1.00	14.39
8	N	TRP	32	24.685	1.112	27.156	1.00	18.61
9	CA	TRP	32	24.896	1.996	28.316	1.00	17.67
10	CB	TRP	32	26.253	1.657	28.999	1.00	18.46
11	CG	TRP	32	26.543	2.508	30.252	1.00	14.22
12	CD2	TRP	32	26.947	3.865	30.325	1.00	16.45
13	CE2	TRP	32	27.044	4.089	31.715	1.00	13.95
14	CE3	TRP	32	27.232	4.916	29.444	1.00	14.91
15	CD1	TRP	32	26.405	1.970	31.509	1.00	19.11
16	NE1	TRP	32	26.722	2.948	32.369	1.00	17.55
17	CZ2	TRP	32	27.417	5.348	32.222	1.00	16.49
18	CZ3	TRP	32	27.602	6.164	29.953	1.00	8.45
19	CH2	TRP	32	27.698	6.373	31.321	1.00	11.56
20	C	TRP	32	24.917	3.414	27.781	1.00	16.08
21	O	TRP	32	24.363	4.325	28.378	1.00	17.69
22	N	ILE	33	25.536	3.534	26.593	1.00	16.72
23	CA	ILE	33	25.591	4.911	26.052	1.00	17.89
24	CB	ILE	33	26.670	5.169	24.944	1.00	20.24
25	CG2	ILE	33	26.790	6.671	24.704	1.00	18.87
26	CG1	ILE	33	28.038	4.571	25.295	1.00	16.21
27	CD1	ILE	33	28.930	4.480	24.013	1.00	24.09
28	C	ILE	33	24.196	5.403	25.732	1.00	18.98
29	O	ILE	33	23.877	6.540	26.194	1.00	18.61
30	N	ARG	36	22.046	6.096	28.836	1.00	20.61
31	CA	ARG	36	22.587	7.077	29.780	1.00	20.93
32	CB	ARG	36	23.940	6.602	30.339	1.00	19.27
33	CG	ARG	36	23.882	5.328	31.146	1.00	20.40
34	CD	ARG	36	23.627	5.619	32.605	1.00	22.27
35	NE	ARG	36	23.511	4.396	33.393	1.00	27.02
36	CZ	ARG	36	23.867	4.298	34.670	1.00	25.93
37	NH1	ARG	36	23.734	3.152	35.315	1.00	26.63
38	NH2	ARG	36	24.330	5.355	35.318	1.00	23.35
39	C	ARG	36	22.702	8.517	29.247	1.00	18.28
40	O	ARG	36	22.703	9.462	30.029	1.00	17.41
41	N	THR	37	22.798	8.697	27.936	1.00	18.97
42	CA	THR	37	22.932	10.050	27.405	1.00	21.02
43	CB	THR	37	24.388	10.371	26.949	1.00	18.78
44	OG1	THR	37	24.793	9.461	25.925	1.00	17.72
45	CG2	THR	37	25.347	10.293	28.084	1.00	21.35
46	C	THR	37	22.048	10.362	26.222	1.00	20.16
47	O	THR	37	21.914	11.534	25.839	1.00	25.43
48	N	CAC	112	30.456	25.709	28.104	1.00	10.38
49	CA	CAC	112	29.270	25.229	27.412	1.00	14.44
50	CB	CAC	112	28.799	23.888	27.980	1.00	17.69
51	SG	CAC	112	29.712	22.439	27.254	1.00	17.65
52	CD	CAC	112	32.183	21.508	28.594	1.00	24.17
53	CE	CAC	112	30.937	22.403	28.616	1.00	21.28
54	OE	CAC	112	30.752	23.125	29.602	1.00	25.29
55	C	CAC	112	28.167	26.294	27.295	1.00	11.81
56	O	CAC	112	27.369	26.232	26.368	1.00	10.19
57	N	LEU	142	35.611	19.985	21.261	1.00	10.22
58	CA	LEU	142	35.860	19.347	22.539	1.00	13.06
59	CB	LEU	142	34.735	19.597	23.555	1.00	12.36
60	CG	LEU	142	34.583	20.999	24.171	1.00	11.62
61	CD1	LEU	142	33.937	20.919	25.543	1.00	5.06
62	CD2	LEU	142	35.940	21.651	24.300	1.00	10.88
63	C	LEU	142	36.175	17.851	22.433	1.00	13.55
64	O	LEU	142	36.786	17.299	23.322	1.00	19.07
65	N	ARG	151	36.295	6.724	29.164	1.00	23.03
66	CA	ARG	151	34.919	6.417	28.730	1.00	23.11
67	CB	ARG	151	34.470	5.004	29.175	1.00	16.86
68	CG	ARG	151	34.348	4.774	30.666	1.00	15.32
69	CD	ARG	151	33.926	3.335	30.928	1.00	7.13
70	NE	ARG	151	33.779	3.086	32.349	1.00	10.71
71	CZ	ARG	151	33.378	1.927	32.869	1.00	3.91
72	NH1	ARG	151	33.268	1.783	34.179	1.00	4.61
73	NH2	ARG	151	33.078	0.930	32.071	1.00	10.10
74	C	ARG	151	33.873	7.478	29.120	1.00	17.49
75	O	ARG	151	33.012	7.828	28.317	1.00	17.71
76	N	GLY	152	34.016	8.044	30.309	1.00	17.52
77	CA	GLY	152	33.070	9.045	30.776	1.00	16.37
78	C	GLY	152	33.062	10.401	30.082	1.00	15.84
79	O	GLY	152	32.246	11.248	30.439	1.00	21.56
80	N	ILE	155	32.443	9.844	25.187	1.00	7.71
81	CA	ILE	155	31.083	9.426	24.707	1.00	12.55
82	CB	ILE	155	30.385	8.425	25.708	1.00	11.77
83	CG2	ILE	155	31.197	7.148	25.866	1.00	11.90
84	CG1	ILE	155	30.158	9.085	27.088	1.00	12.15
85	CD1	ILE	155	29.158	8.276	27.966	1.00	11.79
86	C	ILE	155	30.193	10.622	24.373	1.00	10.55
87	O	ILE	155	29.530	10.593	23.314	1.00	14.21
88	N	ILE	156	30.115	11.601	25.228	1.00	15.15
89	CA	ILE	156	29.284	12.781	24.971	1.00	13.87
90	CB	ILE	156	28.912	13.460	26.383	1.00	18.45
91	CG2	ILE	156	27.632	12.860	26.931	1.00	23.09
92	CG1	ILE	156	30.082	13.252	27.370	1.00	15.34
93	CD1	ILE	156	29.617	12.611	28.714	1.00	19.30
94	C	ILE	156	29.845	13.826	24.026	1.00	13.98
95	O	ILE	156	29.049	14.365	23.211	1.00	9.76
96	N	PHE	157	31.114	14.104	24.000	1.00	10.77
97	CA	PHE	157	31.656	15.152	23.157	1.00	7.33
98	CB	PHE	157	32.859	15.790	23.759	1.00	4.54
99	CG	PHE	157	32.560	16.451	25.090	1.00	7.66
100	CD1	PHE	157	32.946	15.788	26.255	1.00	3.98
101	CD2	PHE	157	31.915	17.650	25.184	1.00	5.65
102	CE1	PHE	157	32.660	16.349	27.491	1.00	9.88
103	CE2	PHE	157	31.630	18.205	26.422	1.00	4.05
104	CZ	PHE	157	32.018	17.536	27.588	1.00	6.80
105	C	PHE	157	31.810	14.851	21.690	1.00	10.70
106	O	PHE	157	32.380	13.859	21.257	1.00	13.03
107	N	LEU	189	34.231	20.663	36.441	1.00	15.69
108	CA	LEU	189	34.309	20.542	34.989	1.00	15.11
109	CB	LEU	189	32.983	20.982	34.350	1.00	10.07
110	CG	LEU	189	32.807	20.922	32.844	1.00	7.51
111	CD1	LEU	189	33.311	19.593	32.263	1.00	10.35
112	CD2	LEU	189	31.343	21.142	32.523	1.00	7.61
113	C	LEU	189	35.464	21.418	34.538	1.00	15.40
114	O	LEU	189	35.452	22.612	34.812	1.00	16.51
115	N	LEU	205	40.306	17.390	29.143	1.00	13.16
116	CA	LEU	205	39.050	17.802	29.770	1.00	15.27
117	CB	LEU	205	37.963	17.874	28.694	1.00	12.62
118	CG	LEU	205	36.505	18.215	29.034	1.00	14.99
119	CD1	LEU	205	35.817	18.527	27.706	1.00	15.12
120	CD2	LEU	205	35.773	17.085	29.762	1.00	11.51
121	C	LEU	205	38.658	16.793	30.846	1.00	15.81
122	O	LEU	205	38.675	15.588	30.594	1.00	20.20
123	N	MET	207	35.792	15.888	34.121	1.00	18.42
124	CA	MET	207	34.419	16.232	34.463	1.00	16.18
125	CB	MET	207	33.555	16.227	33.174	1.00	17.87
126	CG	MET	207	32.024	16.237	33.467	1.00	17.17
127	SD	MET	207	30.990	16.464	32.044	2.00	17.60
128	CE	MET	207	31.340	14.797	31.582	1.00	22.99
129	C	MET	207	33.790	15.238	35.466	1.00	16.62
130	O	MET	207	33.726	14.046	35.222	1.00	18.22
131	N	GLY	209	30.811	14.103	36.169	1.00	12.42
132	CA	GLY	209	29.492	14.040	35.588	1.00	16.72
133	C	GLY	209	28.358	14.011	36.516	1.00	19.06
134	O	GLY	209	27.487	14.883	36.423	1.00	20.59
135	N	ASN	210	28.284	13.037	37.418	1.00	21.24
136	CA	ASN	210	27.150	13.010	38.362	1.00	24.44
137	CB	ASN	210	27.198	11.753	39.171	1.00	25.49
138	CG	ASN	210	27.160	11.958	40.631	1.00	33.50
139	OD1	ASN	210	26.177	11.619	41.309	1.00	34.80
140	ND2	ASN	210	28.217	12.429	41.247	1.00	32.41
141	C	ASN	210	26.970	14.201	39.196	1.00	25.55
142	O	ASN	210	25.858	14.799	39.270	1.00	27.11
143	N	VAL	212	27.967	17.255	38.323	1.00	18.86
144	CA	VAL	212	27.657	18.365	37.397	1.00	19.45
145	CB	VAL	212	28.483	18.363	36.115	1.00	13.66
146	CG1	VAL	212	28.142	19.417	35.091	1.00	10.49
147	CG2	VAL	212	29.921	18.642	36.480	1.00	11.31
148	C	VAL	212	26.176	18.359	36.977	1.00	25.20
149	O	VAL	212	25.455	19.359	36.929	1.00	27.15
150	N	PHE	213	25.738	17.114	36.763	1.00	24.63
151	CA	PHE	213	24.361	16.813	36.336	1.00	25.87
152	CB	PHE	213	24.203	15.287	36.398	1.00	23.74
153	CG	PHE	213	22.788	14.958	36.099	1.00	23.97
154	CD1	PHE	213	22.533	14.398	34.810	1.00	27.08
155	CD2	PHE	213	21.752	14.909	36.974	1.00	22.61
156	CE1	PHE	213	21.275	13.964	34.464	1.00	23.26
157	CE2	PHE	213	20.480	14.482	36.625	1.00	23.27
158	CZ	PHE	213	20.223	13.976	35.335	1.00	21.74
159	C	PHE	213	23.356	17.458	37.319	1.00	26.46
160	O	PHE	213	22.395	18.091	36.945	1.00	28.12
161	N	ALA	216	23.435	21.215	37.390	1.00	21.80
162	CA	ALA	216	22.949	21.675	36.100	1.00	19.74
163	CB	ALA	216	23.464	20.861	34.933	1.00	18.25
164	C	ALA	216	21.440	21.811	36.028	1.00	20.86
165	O	ALA	216	20.936	22.882	35.612	1.00	14.72
166	N	HIS	244	21.005	23.509	25.764	1.00	14.90
167	CA	HIS	244	22.348	23.098	25.390	1.00	17.43
168	CB	HIS	244	23.328	23.551	26.478	1.00	17.97
169	CG	HIS	244	24.644	22.836	26.459	1.00	18.58
170	CD2	HIS	244	25.582	22.714	25.488	1.00	18.43
171	ND1	HIS	244	25.123	22.136	27.546	1.00	18.75
172	CE1	HIS	244	26.295	21.608	27.243	1.00	21.88
173	NE2	HIS	244	26.597	21.944	26.000	1.00	17.34
174	C	HIS	244	22.190	21.563	25.366	1.00	17.94
175	O	HIS	244	21.579	20.979	26.286	1.00	18.08
176	N	ALA	246	23.569	18.461	26.118	1.00	19.92
177	CA	ALA	246	24.594	17.753	26.886	1.00	22.75
178	CB	ALA	246	24.851	18.474	28.207	1.00	20.40
179	C	ALA	246	24.197	16.301	27.174	1.00	25.65
180	O	ALA	246	24.941	15.364	26.869	1.00	27.18
181	N	ASN	247	23.035	16.122	27.793	1.00	26.14
182	CA	ASN	247	22.545	14.795	28.146	1.00	26.38
183	CB	ASN	247	22.964	14.464	29.587	1.00	28.11
184	CG	ASN	247	22.574	13.044	30.019	1.00	32.46
185	OD1	ASN	247	21.552	12.486	29.583	1.00	30.09
186	ND2	ASN	247	23.371	12.470	30.912	1.00	31.19
187	C	ASN	247	21.021	14.827	28.020	1.00	26.38
188	O	ASN	247	20.381	15.783	28.497	1.00	24.78
189	N	ARG	249	18.806	13.418	29.619	1.00	26.17
190	CA	ARG	249	18.082	13.368	30.918	1.00	30.19
191	CB	ARG	249	18.684	12.450	31.892	1.00	35.11
192	CG	ARG	249	20.149	12.514	32.084	1.00	40.00
193	CD	ARG	249	20.737	11.377	33.040	1.00	40.00
194	NE	ARG	249	19.770	10.270	32.981	1.00	40.00
195	CZ	ARG	249	20.131	8.991	32.720	1.00	40.00
196	NH1	ARG	249	19.206	8.005	32.728	1.00	40.00
197	NH2	ARG	249	21.400	8.728	32.469	1.00	40.00
198	C	ARG	249	17.883	14.720	31.505	1.00	30.27
199	O	ARG	249	16.848	15.128	31.999	1.00	29.21
200	N	ILE	250	19.022	15.485	31.317	1.00	31.13
201	CA	ILE	250	18.989	16.891	31.777	1.00	30.26
202	CB	ILE	250	20.417	17.557	31.646	1.00	31.49
203	CG2	ILE	250	20.269	19.060	31.848	1.00	27.57
204	CG1	ILE	250	21.391	16.967	32.703	1.00	27.31
205	CD1	ILE	250	22.804	17.587	32.626	1.00	29.25
206	C	ILE	250	17.878	17.652	31.051	1.00	29.32
207	O	ILE	250	17.014	18.274	31.667	1.00	30.29
208	N	ASN	274	27.325	16.399	22.555	1.00	13.47
209	CA	ASN	274	27.474	17.720	23.155	1.00	13.39
210	CB	ASN	274	27.818	17.530	24.622	1.00	15.56
211	CG	ASN	274	27.960	18.816	25.366	1.00	17.87
212	OD1	ASN	274	28.135	19.881	24.780	1.00	24.67
213	ND2	ASN	274	27.890	18.729	26.689	1.00	19.64
214	C	ASN	274	28.638	18.414	22.458	1.00	14.33
215	O	ASN	274	29.770	17.971	22.613	1.00	12.94
216	N	SER	276	29.549	21.633	22.863	1.00	7.51
217	CA	SER	276	29.823	22.861	23.613	1.00	13.37
218	CB	SER	276	31.354	23.045	23.758	1.00	16.08
219	OG	SER	276	31.709	24.178	24.552	1.00	13.44
220	C	SER	276	29.132	24.114	23.029	1.00	13.89
221	O	SER	276	27.945	24.062	22.700	1.00	11.72
222	N	PHE	304	24.334	25.567	30.088	1.00	14.66
223	CA	PHE	304	25.107	25.471	31.332	1.00	17.36
224	CB	PHE	304	24.396	24.476	32.274	1.00	14.19
225	CG	PHE	304	25.035	24.321	33.630	1.00	14.80
226	CD1	PHE	304	26.179	23.562	33.795	1.00	13.55
227	CD2	PHE	304	24.464	24.909	34.748	1.00	18.11
228	CE1	PHE	304	26.751	23.388	35.053	1.00	13.17
229	CE2	PHE	304	25.024	24.744	36.014	1.00	19.56
230	CZ	PHE	304	26.175	23.977	36.166	1.00	18.61
231	C	PHE	304	26.495	24.936	30.934	1.00	18.48
232	O	PHE	304	26.597	24.072	30.048	1.00	19.82
233	N	GLY	305	27.546	25.411	31.603	1.00	20.16
234	CA	GLY	305	28.889	24.966	31.272	1.00	18.15
235	C	GLY	305	29.950	25.008	32.367	1.00	15.06
236	O	GLY	305	29.701	25.407	33.507	1.00	11.78
237	N	GLY	306	31.145	24.556	31.988	1.00	16.84
238	CA	GLY	306	32.290	24.514	32.875	1.00	16.87
239	C	GLY	306	32.529	25.856	33.525	1.00	19.36
240	O	GLY	306	32.236	26.899	32.934	1.00	17.63
241	N	PHE	487	38.425	26.469	30.807	1.00	13.64
242	CA	PHE	487	37.277	26.474	31.704	1.00	12.94
243	CB	PHE	487	35.953	26.064	31.031	1.00	16.12
244	CG	PHE	487	35.967	24.728	30.332	1.00	10.50
245	CD1	PHE	487	36.055	24.668	28.952	1.00	11.96
246	CD2	PHE	487	35.776	23.548	31.043	1.00	14.59
247	CE1	PHE	487	35.943	23.450	28.275	1.00	11.67
248	CE2	PHE	487	35.665	22.321	30.373	1.00	10.83
249	CZ	PHE	487	35.748	22.283	28.989	1.00	9.41
250	C	PHE	487	37.606	25.574	32.861	1.00	13.75
251	O	PHE	487	38.217	24.529	32.661	1.00	18.53
252	AO6	COA	350	25.886	9.541	33.559	1.00	40.00
253	AP2	COA	350	25.938	8.466	34.779	1.00	40.00
254	AO4	COA	350	25.984	7.033	34.193	1.00	40.00
255	AO5	COA	350	24.688	8.689	35.674	1.00	40.00
256	AO3	COA	350	27.383	8.800	35.491	1.00	40.00
257	AP1	COA	350	27.959	7.998	36.780	1.00	40.00
258	AO1	COA	350	26.887	7.993	37.879	1.00	40.00
259	AO2	COA	350	29.237	8.653	37.296	1.00	40.00
260	AO5*	COA	350	28.201	6.460	36.164	1.00	40.00
261	AC5*	COA	350	27.718	5.279	36.817	1.00	39.18
262	AC4*	COA	350	28.472	4.019	36.378	1.00	37.65
263	AO4*	COA	350	28.702	4.012	34.931	1.00	35.45
264	AC3*	COA	350	29.898	3.856	36.965	1.00	37.54
265	AO3*	COA	350	30.205	2.474	37.178	1.00	40.00
266	AP3*	COA	350	31.518	2.029	38.160	1.00	40.00
267	AO7	COA	350	32.888	2.220	37.337	1.00	40.00
268	AO8	COA	350	31.503	3.018	39.420	1.00	40.00
269	AO9	COA	350	31.296	0.500	38.522	1.00	40.00
270	AC2*	COA	350	30.688	4.469	35.850	1.00	32.65
271	AO2*	COA	350	32.112	4.433	35.932	1.00	24.96
272	AC1*	COA	350	30.098	3.815	34.584	1.00	27.72
273	AN9	COA	350	30.429	4.564	33.382	1.00	20.99
274	AC8	COA	350	30.840	5.878	33.186	1.00	21.31
275	AN7	COA	350	30.992	6.002	31.788	1.00	18.53
276	AC5	COA	350	30.700	4.873	31.234	1.00	12.67
277	AC6	COA	350	30.698	4.501	29.898	1.00	12.21
278	AN6	COA	350	31.039	5.381	28.963	1.00	15.81
279	AN1	COA	350	30.338	3.249	29.672	1.00	17.72
280	AC2	COA	350	30.014	2.442	30.654	1.00	11.38
281	AN3	COA	350	29.997	2.743	31.973	1.00	15.08
282	AC4	COA	350	30.341	3.964	32.268	1.00	15.56
283	PS1	COA	350	27.926	20.647	30.314	1.00	40.00
284	PC2	COA	350	26.439	19.897	31.045	1.00	40.00
285	PC3	COA	350	26.760	18.654	31.835	1.00	40.00
286	PN4	COA	350	26.965	17.518	30.873	1.00	40.00
287	PC5	COA	350	27.350	16.338	31.273	1.00	40.00
288	PO5	COA	350	27.542	15.370	30.476	1.00	40.00
289	PC6	COA	350	27.580	16.199	32.745	1.00	40.00
290	PC7	COA	350	26.255	15.801	33.363	1.00	40.00
291	PN8	COA	350	26.292	14.370	33.634	1.00	40.00
292	PC9	COA	350	26.176	13.440	32.669	1.00	37.37
293	PO9	COA	350	25.948	13.691	31.437	1.00	31.87
294	PC10	COA	350	26.320	11.982	33.151	1.00	38.48
295	PC10	COA	350	26.849	11.940	34.496	1.00	37.07
296	PC11	COA	350	27.172	11.057	32.178	1.00	40.00
297	PC13	COA	350	28.667	11.476	32.189	1.00	40.00
298	PC14	COA	350	26.632	11.101	30.745	1.00	40.00
299	PC12	COA	350	26.933	9.588	32.579	1.00	40.00

[0031] Mutants and Derivatives

[0032] The invention further provides homologues, co-complexes and mutants of the E. coli FabH crystal structure of the invention.

[0033] The term “homologue” means a protein having at least 30% amino acid sequence identity with E. coli FabH or any of its functional domains.

[0034] The term “co-complex” means FabH or a mutant or homologue of FabH in covalent or non-covalent association with a chemical entity or compound.

[0035] The term “mutant” refers to a FabH polypeptide, i.e., a polypeptide displaying the biological activity of wild-type FabH activity, characterized by the replacement of at least one amino acid from the wild-type FabH sequence. Such a mutant may be prepared, for example, by expression of E. coli FabH cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis.

[0036] E. coli FabH mutants may also be generated by site-specific incorporation of unnatural amino acids into the FabH proteins using the general biosynthetic method of C. J. Noren et al, Science, 244:182-188 (1989). In this method, the codon encoding the amino acid of interest in wild-type FabH enzyme is replaced by a “blank” nonsense codon, TAG, using oligonucleotide-directed mutagenesis. A suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid. The aminoacylated tRNA is then added to an in vitro translation system to yield a mutant FabH enzyme with the site-specific incorporated unnatural amino acid.

[0037] Selenomethionine may be incorporated into wild-type or mutant FabH by expression of E. coli FabH-encoding cDNAs in auxotrophic or non-auxotrophic E. coli strains [W. A. Hendrickson et al, EMBO J., 9(5):1665-1672 (1990)]. In this method, the wild-type or mutagenized FabH cDNA may be expressed in a host organism on a growth medium depleted of either natural methionine but enriched in selenomethionine. The location(s) of the Se atom(s) can be determined by X-ray diffraction analysis at three or four different wavelengths. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the enzyme.

[0038] II. Methods of Identifying Inhibitors of the Novel FabH Crystalline Structure

[0039] Another aspect of this invention involves a method for identifying inhibitors of a FabH enzyme characterized by the crystal structure and novel active site described herein, and the inhibitors themselves. The novel E. coli FabH crystalline structure of the invention, or the structure of E. coli FabH homologue, permits the identification of inhibitors of FabH activity. Such inhibitors may be competitive, binding to all or a portion of the active site of FabH; or non-competitive and bind to and inhibit FabH whether or not it is bound to another chemical entity.

[0040] One design approach is to probe the FabH crystal of the invention with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate 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 FabH inhibitor activity [J. Travis, Science, 262:1374 (1993)].

[0041] 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 or with FabH. Thus, the time-dependent analysis of structural changes in FabH during its interaction with other molecules is permitted. The reaction intermediates of FabH can also be deduced from the reaction product in co-complex with FabH. Such information is useful to design improved analogues of known FabH inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the enzyme and enzyme-inhibitor co-complex. This provides a novel route for designing FabH inhibitors with both high specificity and stability.

[0042] Another approach made possible 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 FabH 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 [E. C. Meng et al, J. Comp. Chem., 13:505-524 (1992)].

[0043] Because FabH may crystallize in more than one crystal form, the structure coordinates of FabH, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of FabH. They may also be used to solve the structure of FabH mutant co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of FabH.

[0044] 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 FabH, a FabH mutant, a FabH co-complex, a FabH from a different bacterial species, or the crystal of some other protein with significant amino acid sequence homology to any domain of FabH, may be determined using the FabH structure coordinates of this invention as provided in FIG. 1 and Tables I-III. This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.

[0045] Thus, the FabH structure provided herein permits the screening of known molecules and/or the designing of new molecules which bind to the structure, particularly at the active site or substrate binding site, via the use of computerized evaluation systems. For example, computer modeling systems are available in which the sequence of the FabH, and the FabH structure (i.e., the atomic coordinates, bond distances between atoms in the active site region, etc. as provided by FIGS. 1-2 and Tables I-III herein) may be input. Thus, a machine readable medium may be encoded with data representing the coordinates of FIGS. 1-2 and Tables I-III. The computer then generates structural details of the site into which a test compound should bind, thereby enabling the determination of the complementary structural details of said test compound.

[0046] More particularly, the design of compounds that bind to or inhibit FabH according to this invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with FabH. Non-covalent molecular interactions important in the association of FabH with its substrate include hydrogen bonding, van der Waals and hydrophobic interactions.

[0047] Second, the compound must be able to assume a conformation that allows it to associate with FabH. Although certain portions of the compound will not directly participate in this association with FabH, 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 substrate binding sites of FabH, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with FabH.

[0048] The potential inhibitory or binding effect of a chemical compound on FabH 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 FabH, synthesis and testing of the compound is obviated. However, if computer modelling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to FabH and inhibit using a suitable assay. In this manner, synthesis of inoperative compounds may be avoided.

[0049] An inhibitory or other binding compound of FabH 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 pockets or other areas of FabH.

[0050] One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with FabH and more particularly with the individual binding pockets of the FabH active site or accessory binding sites. This process may begin by visual inspection of, for example, the active site on the computer screen based on the FabH coordinates in FIGS. 1-2 and Tables I-III. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within a binding pocket of FabH. 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.

[0051] Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:

[0052] 1. GRID [P. J. Goodford, “A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules”, J. Med. Chem., 28:849-857 (1985)]. GRID is available from Oxford University, Oxford, UK.

[0053] 2. MCSS [A. Miranker and M. Karplus, “Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method”, Proteins: Structure. Function and Genetics, 11:29-34 (1991)]. MCSS is available from Molecular Simulations, Burlington, Mass.

[0054] 3. AUTODOCK [D. S. Goodsell and A. J. Olsen, “Automated Docking of Substrates to Proteins by Simulated Annealing”, Proteins: Structure, Function, and Genetics, 8:195-202 (1990)]. AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.

[0055] 4. DOCK [I. D. Kuntz et al, “A Geometric Approach to Macromolecule-Ligand Interactions”, J. Mol. Biol., 161:269-288 (1982)]. DOCK is available from University of California, San Francisco, Calif.

[0056] 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 FabH. This would be followed by manual model building using software such as Quanta or Sybyl.

[0057] Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include:

[0058] 1. CAVEAT [P. A. Bartlett 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)]. CAVEAT is available from the University of California, Berkeley, Calif.

[0059] 2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This area is reviewed in Y. C. Martin, “3D Database Searching in Drug Design”, J. Med. Chem., 35:2145-2154 (1992).

[0060] 3. HOOK (available from Molecular Simulations, Burlington, Mass.).

[0061] Instead of proceeding to build a FabH inhibitor in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other FabH 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:

[0062] 1. LUDI [H.-J. Bohm, “The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors”, J. Comp. Aid. Molec. Design, 6:61-78 (1992)]. LUDI is available from Biosym Technologies, San Diego, Calif.

[0063] 2. LEGEND [Y. Nishibata and A. Itai, Tetrahedron, 47:8985 (1991)]. LEGEND is available from Molecular Simulations, Burlington, Mass.

[0064] 3. LeapFrog (available from Tripos Associates, St. Louis, Mo.).

[0065] Other molecular modelling techniques may also be employed in accordance with this invention. See, e.g., N. C. Cohen et al, “Molecular Modeling Software and Methods for Medicinal Chemistry”, J. Med. Chem., 33:883-894 (1990). See also, M. A. Navia and M. A. Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2:202-210 (1992). For example, where the structures of test compounds are known, a model of the test compound may be superimposed over the model of the structure of the invention. Numerous methods and techniques are known in the art for performing this step, any of which may be used. See, e.g., P. S. Farmer, Drug Design, Ariens, E. J., ed., Vol. 10, pp 119-143 (Academic Press, New York, 1980); U.S. Pat. No. 5,331,573; U.S. Pat. No. 5,500,807; C. Verlinde, Curr. Biol., 2:577-587 (1994); and I. D. Kuntz, Science, 257:1078-1082 (1992). The model building techniques and computer evaluation systems described herein are not a limitation on the present invention.

[0066] Thus, using these computer evaluation systems, a large number of compounds may be quickly and easily examined and expensive and lengthy testing avoided. Moreover, the need for actual synthesis of many compounds is effectively eliminated.

[0067] Once identified by the modelling techniques, the FabH inhibitor may be tested for bioactivity using standard techniques. For example, structure of the invention may be used in binding assays using conventional formats to screen inhibitors. One particularly suitable assay format includes the enzyme-linked immunosorbent assay (ELISA). Other assay formats may be used; these assay formats are not a limitation on the present invention.

[0068] In another aspect, the FabH structure of the invention permit the design and identification of synthetic compounds and/or other molecules which are characterized by the conformation of FabH of the invention. Using known computer systems, the coordinates of the FabH structure of the invention may be provided in machine readable form, the test compounds designed and/or screened and their conformations superimposed on the structure of the FabH of the invention. Subsequently, suitable candidates identified as above may be screened for the desired FabH inhibitory bioactivity, stability, and the like.

[0069] Once identified and screened for biological activity, these inhibitors may be used therapeutically or prophylactically to block FabH activity, and thus, bacterial replication.

[0070] The following examples illustrate various aspects of this invention. These examples do not limit the scope of this invention which is defined by the appended claims.

EXAMPLE 1

The Expression of FabH from Escherichia coli in Escherichia coli

[0071] The strategy for the expression of the FabH from Escherichia coli, using Escherichia coli as a host was based on the PCR amplification of the fabH gene and the introduction of suitable restriction sites that allowed the cloning of the fabH-containing DNA fragment in the pET29 expression vector. After the PCR amplification the insert of the resultant recombinant plasmid, (pET29c hereafter), was sequenced to verify the absence of artifacts introduced by the Taq polymerase reaction. Expression was monitored by SDS-polyacrylamide gel analysis.

[0072] A. Bacterial Strains, Plasmids and Medium

[0073] The Escherichia coli strains used were: MAXEfficiency DH10B Competent Cells Genotype: F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara, leu)7697 galU galK λ− rpsL nupG. E. coli cells were grown at 37° C. in Luria Bertani broth (LB). These strains may all be obtained from commercial sources. BL21(DE3) competent cells for protein expression purchased by Novagen. The protocol used to make them electroporation-competent was the one provided by Invitrogen.

[0074] The plasmid used was pET29 [Novagen]. A detailed description of pET29 is provided in FIG. 2. Briefly, pET29 is an expression vector of E.coli which is based on the T7 promoter-driven system and allows the selection of the recombinant clones by kanamycin resistance.

[0075] LB Medium. Per liter:

Bacto-tryptone      10 g
Bacto-yeast extract 5 g
NaCl                5 g

[0076] For plasmid propagation 0.1 mg/ml of kanamycin was added to the medium.

[0077] B. DNA Manipulations

[0078] Plasmid DNA was prepared by the rapid alkaline method (Sambrook et al, 1989). Transformations of E. coli cells were carried out according to the protocol provided with the DH10B or the electroporation method. The plasmids for sequencing were purified using QIAGEN mini-prep kit [QIAGEN]. DNA sequencing was carried out on supercoiled plasmid DNA by the dideoxy chain-termination method using the Thermo Sequenase cycle sequencing kit [ABI, BigDye, Applied BioSystems, USA]. Synthetic oligonucleotides [ordered in-house] were used as primers. Restriction enzymes and T4DNA ligase were obtained from Gibco BRL (Life Technologies) and the experiments were carried out following the instructions provided by the suppliers.

[0079] The fabH gene from E.coli cloned in the pET29 vector was amplified by PCR using the primers:

(5′-TATACATATGTATACGAAGATTATTGGT-3′; SEQ ID
                                 NO: 2)
and:
(5′-ATATGGATCCCTAGAAACGAACCAGCGCGG-3′;. SEQ ID
                                 NO: 3)

[0080] NdeI and BamHI restriction sites were incorporated at the 5′ and 3′ ends respectively of each primer to facilitate ligation of the amplified DNA into vectors. Plasmid DNA (480 ng) was amplified in 100 ul of PCR mixture containing 200 uM deoxynucleotide triphosphates (dNTPs), 0.20 mM oligonucleotide primers, the manufacturer's buffer and 2.5 U of pfu (Stratagene). The following cycling parameters were used:

[0081] 94° C. 4 min

[0082] 94° C. 1 min, 55° C. 40 sec, 72° C. 1 min (30 cycles)

[0083] 72° C. 2 min

[0084] 4° C.

[0085] Polymerase chain reaction (PCR) was performed using the GeneAmp, PCR System 2400 [Perkin Elmer Cetus Co]. PCR-amplified DNA fragments were purified using Qiaquick PCR Purification kit for Rapid Purification of DNA Fragments [Qiagen].

[0086] C. Cloning of the fabH Gene of E. coli in the Expression Vector pET29 of E. coli.

[0087] The cloning strategy is shown in FIG. 2. PCR amplification of the fabH gene from E. coli using the primers AKK2 and AKK3 resulted in a DNA fragment of about 960 bp. This fragment was purified with Qiaquick PCR purification kit protocol (Qiagen) digested with NdeI and BamHI, purified, ligated overnight to the NdeI and BamHI sites of already digested vector pET29 to obtain the recombinant plasmid pET29c. The ligation mix was used to transform E. coli DH10B competent cells. The construction of pET29c was initially confirmed by restriction analysis of the plasmid purified from the transformants. The amplification with Taq DNA polymerase made the sequencing of the fabH of pET29c an obligatory step to confirm that no changes were introduced due to the low fidelity of this enzyme. Sequence analysis was accomplished by using T7 promoter and terminator primers. The sequencing of both strands showed that no artifacts had been introduced during the amplification process.

[0088] D. Small-Scale Production of FabH from E. coli in E. coli

[0089] The plasmid pET29c and the negative control pET29 (vector without insert) were used to transform the E. coli BL21(DE3) host strain. Single clones of BL21(DE3): pET29c cells were grown overnight at 37° C. in 2 ml of LB medium in the presence of 0.1 mg/ml kanamycin. The cells were then diluted 100-fold in 10 ml LB with kanamycin. When the cultures reached a value of 0.5 at OD600 the fabH expression was induced by addition of isopropyl-thio-galactoside (IPTG) at 0.5 mM of final concentration. After this induction 2 ml samples were taken at different times (1 and 2 hours). The cells were harvested in a microfuge for 3 min, the pellets were washed with 20 mM Tris-HCl pH 8, 1 mM PMSF and resuspended in 100 ul of SDS-PAGE gel-loading buffer. The cells were broken by sonication (15 seconds). The samples were then boiled for 10 minutes and after one spin, 10 ml fractions were analyzed by SDS-PAGE according to the methods of Laemmli [U. K. Laemmli, Nature 227, 680-685 (1970)]. The 4-12% polyacrylamide gels [NOVEX] were stained with Coomassie blue. As shown in FIG. 3 good expression levels were detected from the early stages after induction with IPTG. The evidence was the presence of a prominent band (lanes 2, 4 and 6 in FIG. 3) that was in good agreement with the Mr predicted from the primary sequence. The FabH protein has a theoretical molecular weight of about 33,514 Da.

EXAMPLE 2

Large Scale Growth and Purification of FabH

[0090] A. Large Scale Growth

[0091] A 4 liter fermentation of E.coli BL21(DE3): pET29c was carried out in Luria Bertani medium (LB), containing 100 ug/ml kanamycin. The 8×500 ml flasks were inoculated at 1% (v/v) from an overnight secondary seed culture in single strength LB medium, containing 100 mg/ml kanamycin. The flasks were incubated at 37° C., agitated at 250 rpm in a benchtop shaker. The OD at 600 nm was monitored, and at 0.5 absorbance units, FabH expression was induced with the addition of isopropyl-thiogalactosidase to 0.5 mM and the cells harvested by centrifugation in a Sorval CSA rotor, 2 hours post induction. A total of 20 grams of cell paste was recovered.

[0092] LB Medium, per liter, contains the following components. The medium was supplied by the in-house laboratory.

[0093] Single Strength

Bacto Tryptone      10 g
Bacto Yeast Extract 5 g
Sodium Chloride     5 g

[0094] B. Purification

[0095] 1) Lysis

[0096] 12.5 g of cells of E. coli overexpressing E. coli FabH obtained as described above, were resuspended in 140 ml of 20 mM Tris pH 7.9, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1 mM PMSF (buffer A). Lysozyme ( Sigma Chemicals: hen egg) was added to a final concentration of 1 mg/ml. Cells were incubated at 37° C. for 20 min. The cells were then lysed by sonication in an ice water bath (4×30 sec). DNAse (Sigma; bovine pancreas type 1) was added along with MgCl2 and held at 37° C. for 5 minutes. The solution was centrifuged in a Beckman JA-HS centrifuge at 14,000 g for 60 minutes using a Beckman JA-14 rotor.

[0097] 2) Anion Exchange

[0098] All chromatography was performed on a Pharmacia chromatography system, fitted with a UV detector (Pharmacia, Monitor UV-1). UV (at 280 nm) was monitored during all operations. All operations were performed at 4° C.

[0099] The supernatant from 1) was loaded onto a Q-Sepharose high performance (Pharmacia) column of 50 ml packed into a Pharmacia XK-26 column. The column was equilibrated with buffer A prior to loading. The column was then washed with buffer A (250 ml) at 4 ml/min, and eluted with a linear gradient of buffer A to 1M NaCl in buffer A over 80 minutes at 4 ml/min. The eluate was fractionated into 8 ml fractions using a Pharmacia FRAC 200.

[0100] The eluted fractions were assayed for FabH activity by measurement of incorporation of [14C]Acetyl-CoA to Malonyl-ACP and , and for protein by the Bradford method. Active fractions were analyzed by reducing SDS-PAGE (NOVEX, NuPAGE Bis-Tris 4-12% gradient gel). Active fractions pooled together and dialyzed against Buffer A.

[0101] 3) Anion Exchange Chromatography

[0102] The dialyzed material was loaded onto a MonoQ column equilibrated with buffer A (Pharmacia, 5/5). The column was washed with 20 ml of the equilibration buffer until 280 nm absorbance returned to base line and then eluted with a linear gradient of equilibration buffer to buffer A over 90 minutes at 0.5 ml/min. Fractions were pooled together, collected, assayed for FabH activity.

[0103] 4) Hydroxyapatite/Buffer Exchange

[0104] Eluted fractions are collected (1 ml fraction) and assayed for FabH activity and protein. Active fractions are pooled and the volume was doubled with Buffer B [20 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM DTT and 10% glycerol] to reduce the salt concentration in half. The active eluate was loaded in a hydroxyapatite column and eluted with 0.5 M Potassium Phosphate pH 7.4. The active enzyme was buffer exchanged with 20 mM Tris pH 7.4, 50 mM NaCl 2 mM DTT. This product was greater than 97% purity by SDS PAGE and the activity showed an overall process yield of 60% from 1). N-terminal amino acid analysis confirmed identity.

EXAMPLE 3

Fermentation, Purification and Characterization of Seleno-methionine derivative of Escherichia coli FabH

[0105] A. Fermentation

[0106] To obtain soluble selmet-FabH for purification and crystallization studies, E. coli strain BL21 (DE3) was transformed with pET29c FabH. 50 ul of the seed culture expressing FabH gene product was inoculated into 100 ml of Luria broth, containing kanamycin (50 ug/ml) and glucose (0.6%). On reaching target density of 2 OD, the cells from the seed culture were isolated by centrifugation, resuspended in 100 ml of M9 minimal medium containing 1 mM CaCl2, 1 mM MgSO4, kanamycin ( 50 ug/ml) and glucose (0.6% w/v). The resuspended pellets were then added to 900 ml of the same medium and the cells were grown at 37 C to mid-log phase, A600 of 1.5, at which point lysine, phenylalanine, threonine at 100 mg/l each, and selenomethionine, isoleucine, leucine, and valine at 50 mg/l were added. The culture was shaken for 15 minutes, and then induced with 0.5 mM isopropyl b-D-thiogalactopyranoside (IPTG). The culture was grown for 13 hours, and harvested by centrifugation (speed). 5 ml aliquots were taken prior to and during induction to monitor the expression of selenomethionine FabH. 12 g of cell paste (wet wt) was recovered from 5L. In addition, to compare the expression of selenomethionine derivative to that of wt FabH, a one 1 culture was prepared under identical conditions except that the cells were grown in LB media.

[0107] B. Purification

[0108] All lysis and purification steps were carried out using degassed buffers in a cold room or on ice. 4.5 g of E. coli cells over expressing Fab H were resuspended in 50 ml of 20 mM Tris, 50 mM sodium chloride, 10% glycerol, 0.2 mM PMSF, 2 mM DTT, pH 7.9 (buffer A). Cells were lysed twice at 10,000 psi using Microfluidizer (Microfluidics Corporation, Mass.). Cell debris was removed by centrifugation (Sorvall RC-5B) at 35,000 g for 30 min. The supernatant was applied to a 2.6×4 cm Source Q column (Pharmacia) equilibrated in buffer A. The column was washed with 10 column volumes of buffer A, and eluted with a 10 column volume gradient of 0 to 1.0 M NaCl in buffer A. Eluted fractions were analyzed by 10% SDS-PAGE under reducing conditions. Fab H eluted at 0.2-0.25 M NaCl. Fab H containing fractions were pooled and applied to a 2.6×6 cm Hydroxyapatite column (Bio-Rad, Type I, 40 u) equilibrated in buffer A. The column was eluted with a 30 column volume linear gradient of buffer A to 400 mM potassium phosphate, 10% glycerol, 2 mM DTT. Fab H, which eluted at 80-180 mM potassium phosphate, was diluted 1:2 with 50 mM Tris, 200 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5 (buffer B) and applied to a 1.6×7.5 cm Blue Sepharose column (Pharmacia) equilibrated in buffer B. The column was eluted with buffer B containing 1 M NaCl. Blue Sepharose eluted Fab H fractions were next applied to a 2.6×60 cm Superdex 200 size exclusion column (Pharmacia) equilibrated in 20 mM Tris, 50 mM NaCl, 2 mM DTT, pH 7.4. A total of 23 mg of Fab H was recovered which was concentrated to 13 mg/ml using Amicon 3 filtration device.

[0109] C. Characterization

[0110] i). Mass Spectoscopy

[0111] Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) data were obtained on a PerSeptive Biosystems Voyager RP laser desorption time-of-flight mass spectrometer. Protein samples were prepared for analysis by diluting analyte 1:5 with 3,5-Dimethoxy-4-hydroxy-cinnamic acid (10 mg/ml in 2:1 0.1% trifluoroacetic acid/acetonitrile) for a final concentration of 1-10 pmol/ul. Bovine Beta lactoglobulin A (Sigma) was included as an internal calibrant (MH+18364 Da). Desorption/ionization was accomplished using photon irradiation from a 337-nm pulsed nitrogen laser and 30-keV accelerating energy. Spectra were averaged over ca. 100 laser scans.

[0112] The predicted molecular mass for native FabH is 33516 Da. MALDI-MS analysis of the selenomethionyl incoporated FabH protein construct provided a mass of 33,889 Da. This is in close agreement with the predicted +375Da shift in mass expected for the sulfur to selenium side-chain substitution of eight methionine residues within the protein (33,891 Da theoretical).

[0113] ii). N-terminal Sequence Analysis

[0114] Sequence analysis was performed on a Hewlett-Packard G1000A N-terminal Protein Sequencer with on-line PTH identification using an HP1100 HPLC. Samples were applied directly to biphasic sequencing cartridges and standard 3.1 sequencing method cycles were used.

[0115] N-terminal sequencing results showed negligible native methionine in the first residue. Instead, a unique PTH (phenylthiohydantoin) derivative was observed which eluted 1.6 minutes later than PTH-methionine, and did not coelute with any natural PTH-amino acid derivatives. The increase in hydrophobicity is consistent with the direct detection of the PTH-selenomethionyl amino acid derivative.

[0116] D. Measurement of β-ketoacyl-ACP Synthase III Activity.

[0117] The enzyme catalyses the condensation of acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP. The reaction can be described by three distinct steps: (a) the acyl group of acetyl-CoA is transferred to the active site cysteine resulting in a acyl-enzyme thioester; (b) carbanion formation by the decarboxylation of malonyl-ACP; and (c) carbon-carbon bond formation by nucleophillic attack of the carbanion onto the carbonyl carbon atom of the acyl-enzyme thioester to yield the acetoacetyl-ACP product.

[0118] This reaction can be assayed in order to characterize the enzyme or identify specific inhibitors of its activity in two ways:

[0119] (1) Radiolabeled acetoacetyl-ACP formation can be specifically determined using [3H]-acetyl-CoA and malonyl-ACP. The [3H]-acetyl-CoA substrate is soluble in 10% TCA while the resulting [3H]-acetoacetyl-ACP is not. A reaction mixture containing 100 mM sodium phosphate buffer pH7.0, 1 mM DTT, 34 uM acetyl-CoA, 0.15 uM [3H]-acetyl-CoA (specific activity 25 Ci/mmol), and 7 uM malonyl-ACP is prepared and transfered to a microtiter plate with or without inhibitors already added. The enzyme is added last to start the reaction and the plate is incubated at 37 degrees C. Ten percent TCA is added to stop the reaction, and then 50 ug of BSA as a carrier. Stopped reactions are filtered and washed 2 times with 10% TCA on Wallac GF/A filtermats using a TomTec harvester. The filtermats are dried at 60 degrees C and the radioactivity quantified using Wallac Betaplate scintillation cocktail and a Wallac Microbeta 1450 liquid scintillation counter. IC50s are generated using Grafit 4.0 software and solved using the equation v=Vmax/(1+I/IC50).

[0120] (2) FabG coupling can also be used to measure FabH production of acetoacetyl-ACP by following NADPH consumption at 340 nm. FabG (β-ketoacyl-ACP reductase) will specifically reduce the C3 carbonyl of acetoacetyl-ACP to form β-hydroxybutyryl-ACP. This reduction requires the conversion of NADPH to form NADP+ which can be monitored by following the optical density at wavelength 340 nm.

[0121] (3) FabD coupling is an available assay option in the absence of purified malonyl-ACP. FabD (Malonyl-CoA:ACP transacylase) is responsible for malonic acid transfer from malonyl-CoA to ACP to form malonyl-ACP. This activity can be exploited by applying the techniques described in (1) above together with de novo malonyl-ACP from the FabD reaction.

[0122] E. Ligand Binding to FabH.

[0123] It is also possible to define ligand interactions with FabH in experiments that are not dependent upon enzyme catalyzed turnover of substrates. This type of experiment can be done in a number of ways:

[0124] (1) Effects of Ligand Binding Upon Enzyme Intrinsic Fluorescence (e.g. of Tryptophan). Binding of either natural ligands or inhibitors may result in enzyme conformational changes which alter enzyme fluorescence. Using stopped-flow fluorescence equipment, this can be used to define the microscopic rate constants that describe binding. Alternatively, steady-state fluorescence titration methods can yield the overall dissociation constant for binding in the same way that these are accessed through enzyme inhibition experiments.

[0125] (2) Spectral Effects of Ligands. Where the ligands themselves are either fluorescent or possess chromophores that overlap with enzyme tryptophan fluorescence, binding can be detected either via changes in the ligand fluorescence properties (e.g. intensity, lifetime or polarization) or fluorescence resonance energy transfer with enzyme tryptophans. The ligands could either be inhibitors or variants of the natural ligands.

[0126] (3) Thermal Analysis of the Enzyme:Ligand Complex. Using calorimetric techniques (e.g. Isothermal Calorimetry, Differential Scanning Calorimetry) it is possible to detect thermal changes, or shifts in the stability of FabH which reports and therefore allows the characterization of ligand binding.

EXAMPLE 3

Crystallization of E. coli Wild-Type and Selenomethionine Mutant of FabH

[0127] A. Crystallization

[0128] All crystals were grown at room temperature using the sitting-drop vapor diffusion method. The drop solution was always a 1:1 mixture of the protein sample and the well solutions. For the crystal form 1 of the wild-type protein, the well solution contained 0.1 M HEPES buffer at pH 7.5 and 20% PEG8000. For the crystal form 2 of the selenomethionine mutant protein in complex with acetyl-CoA, the well solution contained 0.05 M Bis-Tris propane buffer at pH 7.0, 0.1 M MgCl2 and 14% PEG6000. Crystals grew overnight and are approximately 0.1 to 0.2 mm in sizes.

[0129] B. X-ray Diffraction Characterization

[0130] All crystals were frozen in liquid nitrogen streams before their characterization using synchrotron X-ray radiation. Diffraction data for the apo form 1 crystal was collected to 2.0 Å resolution. The data is 97.1% complete and 6 fold redundant with a merging R- factor of 7.7%. The crystal belongs to the orthorhombic spacegroup P212121, with cell dimensions a=63.1, b=65.1 and c=166.5 Å. For the Se-Met protein in complex with acetyl-CoA, data were collected at three different wavelengths: 0.9789, 0.9785 and 0.9414 Å. The three data set were of nearly identical quality, with about 80% completion, 6-fold redundancy, 8.5% merging R-factor, and 1.9 Å resolution. The form 2 crystal belongs to the tetragonal spacegroup P41212, with a=b=72.4 and c=102.8 Å.

[0131] C. Structure Solution

[0132] The crystal structure of the Se-Met E. coli FabH mutant in complex with acetyl-CoA was solved to 1.9 Å resolution using the MAD phasing technique with the data sets collected at three different wavelengths and the program SOLVE (Terwilliger & Berendzen, 1999, Acta Cryst. D55, 849-861). All eight Se-Met were located by SOLVE. The overall MAD phasing figure of merit was 0.6 to 1.9 Å resolution, and the overall Z score was as high as 148. The resulting electron density map was of very high quality. The structure of the apo enzyme (crystal form 1) was solved with the molecular replacement method using the acetyl-CoA complex structure as the search model. This crystal form had a FabH dimer in the asymmetric unit, and the R-factor of the solution was only 33%. Two-fold averaged map was then calculated and used for model building.

[0133] D. Model Building and Refinement

[0134] The electron density for the acetyl-CoA complex was very clear and a structure model for the whole FabH protein, the bound acetyl group and CoA, as well as 98 solvent molecules were built in the first round. Standard structural refinement protocols and manual model building led to the current model, which has an R-factor of 27% to 1.9 Å resolution. The model for the apo FabH structure was also built readily, and refined to an R-factor of 18.9% (Rfree of 24.4%) to 2.0 Å resolution. Both models have excellent geometry and do not have any outliers in the Ramanchandran plot, indicating high quality of the atomic coordinates, which contain an estimated error of less than 0.3 Å.

[0135] This invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. The disclosures of the patents, patent applications and publications cited herein are incorporated by reference in their entireties.

Claims

1. A composition comprising a E. coli FabH in crystalline form.

2. The composition according to claim 1 wherein said FabH is a dimer.

3. The composition according to claim 1 wherein said FabH comprises an active site cavity formed by amino acids comprising Cys112, His244 and Asn274

4. The composition of claim 1 wherein said FabH is a E. coli FabH.

5. The composition of claim 3 wherein said FabH is characterized by the coordinates selected from the group consisting of the coordinates of FIGS. 1-2 and Tables I, II, and III.

6. A E. coli FabH crystal.

7. A selenomethionine mutant crystal of a E. coli FabH.

8. An isolated, properly folded FabH molecule or fragment thereof having a conformation comprising the protein coordinates of FIGS. 1-2 and Tables I, II, and III.

9. The molecule according to claim 8 wherein said molecule is a dimer, wherein each monomer is characterized by two similar domains having core of five β-strands, each containing flanking helices, strands and loops, as illustrated in FIG. 3.

10. The molecule according to claim 8 wherein said molecule is a dimer characterized by the dimer interface of FIG. 3.

11. The molecule according to claim 10 which is E. coli FabH.

12. A peptide, peptidomimetic or synthetic molecule which interacts competitively or non-competitively with the active site of a FabH of claim 1.

13. A method of identifying an inhibitor compound capable of binding to, and inhibiting the enzymatic activity of, a E. coli FabH, said method comprising: introducing into a suitable computer program information defining an active site conformation of a E. coli FabH molecule comprising a conformation defined by the coordinates of FIGS. 1-2 and Tables I, II, and III, wherein said program displays the three-dimensional structure thereof; creating a three dimensional structure of a test compound in said computer program; displaying and superimposing the model of said test compound on the model of said active site; assessing whether said test compound model fits spatially into the active site; incorporating said test compound in a biological activity assay for a FabH characterized by said active site; and determining whether said test compound inhibits enzymatic activity in said assay.

14. The method according to claim 13 wherein said FabH molecule is a dimer, wherein each monomer is characterized by two similar domains having core of five β-strands, each containing flanking helices, strands and loops, as illustrated in FIG. 3.

15. A method of identifying an inhibitor compound capable of binding to, and inhibiting the enzymatic activity of, a E. coli FabH, said method comprising: introducing into a suitable computer program information defining an active site conformation of a FabH molecule comprising a conformation defined by the coordinates of FIGS. 1-2 and Tables I, II, and III, wherein said program displays the three-dimensional structure thereof; creating a three dimensional structure of a test compound in said computer program; displaying and superimposing the model of said test compound on the model of said active site; assessing whether said test compound model fits spatially into the active site; incorporating said test compound in a biological activity assay for a FabH characterized by said active site; and determining whether said test compound inhibits enzymatic activity in said assay.

16. The method according to claim 15 wherein said FabH molecule is a dimer, wherein each monomer is characterized by two similar domains having core of five -strands, each containing flanking helices, strands and loops, as illustrated in FIG. 3.

17. A peptide, peptidomimetic or synthetic molecule identified by the method of claim 13 or 15.

18. A method for solving a crystal form comprising using the structural coordinates of a E. coli FabH crystal or portions thereof, to solve a crystal form of a mutant, homologue or co-complex of said FabH by molecular rearrangement.

19. A method of drug design comprising the step of using the structural coordinates of a E. coli FabH crystal to computationally evaluate a chemical entity for associating with the active site and substrate binding sites of E. coli FabH.

20. The method of drug design according to claim 19 comprising the step of using the structure coordinates of E. coli FabH to identify an intermediate in a chemical reaction between said FabH and a compound with is a substrate or inhibitor of said enzyme.

21. The method according to claim 20, wherein said entity is a competitive or non-competitive inhibitor of a E. coli FabH.

22. The method of drug design according to claim 19, using the structure of a FabH homologue that has similar amino acid identities as well as spacial arrangements as those of E. coli FabH listed in Tables I-III.

23. The method of drug design according to claim 20 using the structure of a FabH homologue that has similar amino acid identities as well as spacial arrangements as those of E. coli FabH listed in Tables I-III.

24. The method of drug design according to claim 21 using the structure of a FabH homologue that has similar amino acid identities as well as spacial arrangements as those of E. coli FabH listed in Tables I-III.

25. The method according to claim 19 wherein said structure coordinates comprise the coordinates of FIGS. 1-2 and Tables I, II, and III.

26. The method according to claim 20 wherein said structure coordinates comprise the coordinates of FIGS. 1-2 and Tables I, II, and III.

27. The method according to claim 21 wherein said structure coordinates comprise the coordinates of FIGS. 1-2 and Tables I, II, and III.