JP2003505014A - Novel FabH enzyme, composition capable of binding to the enzyme, and method of using the same - Google Patents

Abstract

  (57) [Summary] The present invention identifies a novel E. coli FabH crystal structure. Also disclosed are methods for identifying inhibitors of these enzymes and / or active sites, and inhibitors identified by these methods.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a method for identifying a novel enzyme active site and designing and selecting an inhibitor of the active site.

Prior Art The pathways for saturated fatty acid biosynthesis are very similar in prokaryotes and eukaryotes. However, its biosynthetic mechanism is completely different. Vertebrates have type I fatty acid synthase (FAS) and all of their enzymatic activity is encoded on one multifunctional polypeptide in which the mature protein is a homodimer. Acyl carrier protein (
ACP) is an integral part of the complex. In contrast, in most bacterial and plant FAS (type II), the reaction is each catalyzed by a distinct monofunctional enzyme,
ACP is a distinct protein. Mycobacteria are unique in that they have both Type I and Type II FAS. Therefore, bacterial systems are likely to be selectively inhibited by broad-spectrum antibacterial agents (Rock, C. & Cronan, J., Biochimi.
ca et Biophysica Acta 1302, 1-16 (1996); Jackowski, S. In Em.
erging Targets in Antibacterial and Antifungal Chemotherapy, J. Sutcliffe
& N. Georgopapadakou, Chapman & Hall, New York (1992); Jackowski, S.
J. Biol. Chem. 264, 7624-7629 (1989)).

The first step in the biosynthetic cycle is to convert malonyl-ACP to acetyl-CoA and F.
It is a step of condensing using abH. Prior to this, malonyl-ACP is synthesized from ACP and malonyl-CoA using FabD, malonyl CoA: ACP transcyclase. In a subsequent round, malonyl-ACP is condensed with growing chain acyl-ACP (FabB and FabF, synthases I and II, respectively). The second step in the extension cycle is the ketoester reduction by NADPH-dependent β-ketoacyl-ACP reductase (FabG). Then, it was subjected to dehydration with β-hydroxyacyl-ACP dehydrase (either FabA or FabZ) to obtain trans-2-enoyl-ACP, which was sequentially treated with enoyl-AC.
Converted to acyl-ACP by P reductase (FabI). Addition of two carbon atoms / cycle in a further round of this cycle yields essentially palmitoyl-ACP, which is then terminated primarily by feedback inhibition of FabH and I by palmitoyl-ACP (Heath Et al., J. Biol. Ch
em. 271, 1833-1836 (1996)).

Cerulenin and thiolactomycin are potent and selective inhibitors of bacterial fatty acid biosynthesis. Extensive experiments with these inhibitors have revealed that this biosynthetic pathway is essential for bacterial survival. None of the antibiotics on the market target fatty acid biosynthesis, and thus the known mechanism of antibiotic resistance does not appear to render the new antibiotic inactive. Demand for a series of novel compounds, such as FabH-targeted antibiotics, has not yet been fully met. FabH enzymes are of interest as potential targets for antibacterial agents. Novel FabH enzyme, which enables the identification and structure-based design of inhibitors, which are useful in the treatment or prevention of diseases, especially those caused by bacteria sharing a catalytic domain with the domain of the invention. There is a requirement for active sites and catalytic pathways.

DISCLOSURE OF THE INVENTION In one aspect, the present invention provides a novel FabH enzyme active site crystal form. In another aspect, the invention provides catalytic residues Cys112, His244.
And novel FabH compositions characterized by Asn274. In yet another aspect, the invention provides novel FabH compositions characterized by an active site of 33 amino acid residues, including catalytic residues. In yet another aspect, the invention provides a method of identifying an inhibitor of a composition as described above, which provides the structural coordinates of the invention to a computerized modeling system and binds to the structure. A method is provided that comprises identifying compounds and screening the identified compounds for biological activity of FabH inhibitors. In a further aspect, the invention provides an inhibitor of the catalytic activity of a composition having a catalytic domain as described above. Another aspect of the invention includes an instrument-readable medium encoded with data representing the coordinates of the three-dimensional structure of a FabH crystal. Other aspects and advantages of this invention are further described in the following detailed description of the preferred embodiments of this invention.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a novel E. coli FabH crystal structure, a novel FabH active site, and its crystal form and active site, which are used as active sites of FabH enzyme. FabH inhibitor compounds (peptides, characterized by their ability to competitively inhibit binding)
Peptidomimetics or synthetic compositions) are provided. Further, in the present invention, a novel FabH crystal structure complexed with an acetyl-CoA substrate, as well as Fa
Identification of acetyl-CoA interacting residues in bH is also provided.

I. Three-dimensional structure of novel FabH crystal The present invention provides a novel FabH crystal structure based on E. coli FabH.
The amino acid sequence of FabH is provided in Table 1 as SEQ ID NO: 1.

[0008] 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 Leu 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

As shown herein, this crystal structure is a tightly bound FabH dimer. Each monomer has two structural domains: the N-terminal domain (residue 1 of SEQ ID NO: 1).
-170) and a C-terminal domain (residues 171-317 of SEQ ID NO: 1). The two domains are similar throughout their folds, each containing a 5-strand β-sheet sandwiched between α-helices and covered with another β-sheet, α-helix and loops. To do. The structural similarity between the two domains of the protein indicates that FabH is probably derived 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. The core structure of E. coli FabH has some similarities to the structure of FabF (Huang et al., EMBO J. 17, 1183-1191 (1998)), but there are major differences in the atomic position of the core β-strand. The structure outside the β-strands is quite different. FabH and Fab
The amino acid sequence identity between F is less than 20%, which is likely to be a major difference. Therefore, the crystal structure of E. coli FabH is novel.

As mentioned above, E. coli FabH is a dimer and each monomer contains an active site. Dimer formation is essential for FabH activity because the active site of the monomer contains at least Phe87, another monomer of the dimer. The present invention is E. coli Fab
It provides both crystalline monomeric and dimeric structures of H. Inhibitors that disrupt the dimer or interact with the dimer are another target for designing and selecting antimicrobial agents. In the present invention, the crystal structure of E. coli FabH was resolved at 2.0Å (crystal form 1), and the selenomethionine mutant protein complexed with acetyl-CoA was measured at 1.9Å (crystal form 2). Its structure was determined using MAD farsing and molecular replacement methods and refined to 18.9% and 27% R-factor, respectively.

Further refinement of the atomic coordinates will change the numbers in FIGS. 1-2 and Tables I-III, and refinement of the crystal structure derived from another crystalline form will result in a new set of coordinates. Will be obtained. However, the distances and angles shown in Table II will retain identities within experimental error, and the relative conformations of residues in the active site will retain identities within experimental error. Let's do it. For example, two independently measured monomers of crystalline form 1 and one monomer of crystalline form 2 do not have the same coordinate values, but the structures of these three monomers are very similar. It has a structure and the spatial correlation between amino acid residues is considered to be the same within experimental error. In fact, the present inventors have found that the structure that can be superposed on the FabH structure in the range where the error of the root mean square at the α-carbon atom is smaller than 1.5Å has structurally close homology, If the mean square root error is the same over all atoms of a protein, they are considered to have the same structure. FIG. 1 provides the atomic coordinates of the E. coli FabH dimer containing 634 amino acids. FIG. 2 provides the atomic coordinates of an E. coli FabH monomer containing 317 amino acids complexed with acetyl-CoA. The FabH enzyme is characterized by an active site that preferably contains binding sites for the first substrate acetyl-CoA and the second substrate malonyl-ACP. The catalytic residues in FabH are Cys163, His3 in FabF.
03 and His340, while Cys112, His244 and Asn274. The difference in catalytic residues is that the amino acid identity (His340
To Asn274), but also in its relative spatial arrangement. FabH Cys112 and Asn274 can successfully overlay FabF Cys163 and His340, while HabH His244
Occupies a completely different position from His303 of FabF. This indicates that the catalytic mechanisms of the two enzymes are completely different. The crystal structures described herein were elucidated with and without acetyl-CoA. Catalytic Cys112 was covalently acetylated and the product CoA was identified as still bound to the active site. The bound CoA allows identification of the length, size and shape of the active site cavity for successful binding to the β-mercaptoethylamine-pattainate arm of CoA. The structure of the acetyl-CoA complex also revealed all the important residues identified as the series of 33 amino acid residues listed in Table I, which interact with CoA and define the active site. did. For example, the adenine portion of CoA is sandwiched between the side chains of Arg151 and Trp32. The structure of the present invention is determined in the absence of malonyl-ACP. However, the same acetyl-CoA binding cavity should also bind malonyl-ACP, as their active site binding regions are very similar and there is no apparent alternative entrance to the active site. Moreover, the surface of FabH molecules is generally negatively charged, while the region immediately outside the active site cavity is positively charged. This surface is mainly composed of three α-
It contains helices (30-37, 209-231 and 248-258) and contains many positively charged amino acids (Arg36, Arg40, Lys214, Hi).
s222, Arg235, Arg249. Lys256. Lys257). Acyl carrier protein (ACP) is known to be extremely acidic or negatively charged, so it is reasonable to assume that this surface is the ACP binding surface.

Table I provides the atomic coordinates at the active site of the apoE.coli FabH structure (of crystalline form 1). Solvent molecules are omitted for clarity, but can be seen in FIG. Residue 487 is Phe87 from another monomer.

[0013] Table I atom residues 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

[0014]     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

[0015]    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

[0016]    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

[0017]    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

[0018]   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

[0019]   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

[0020]   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

[0021]   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

[0022]   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

Table II shows the distance (D) between the atoms of the active site residues that are within 5.0 Å of each other as defined by Table I.

Table II Distance between 1 atom and 2 atoms (D =) 28CA 151CD 4.796 28CB 33CD1 4.204 28CB 151CD 4.292 28CB 33CG1 4.398 28CB 151CG 4.703 28OG1 33CG1 3.472 28OG1 33CD1 3.709 28OG1 151CD 3.743 28OG1 32CE3 3.902 28OG1 151CG 4.159 28OG1 32CZ3 4.306 28OG 155CG2 4.418 28OG1 32CD2 4.776 28OG1 33CB 4.930 28OG1 151NE 4.962 28CG2 33CD1 3.435 28CG2 33CG1 4.093 32N 33N 2.785 32N 33CA 4.198 32N 33C 4.728 32N 33CB 4.967

[0025]   32CA 33N 2.428   32CA 33CA 3.808   32CA 33C 4.422   32CA 33CB 4.890   32CB 33N 3.133   32CB 33CA 4.454   32CG 33N 3.849   32CG 33CA 4.892   32CD2 33N 3.941   32CD2 36CB 4.506   32CD2 36CD 4.511   32CD2 33CA 4.602   32CD2 36NE 4.722   32CE2 36CD 3.747   32CE2 36NE 3.804   32CE2 36CZ 4.270   32CE2 36CB 4.465   32CE2 36NH2 4.640   32CE2 36CG 4.706   32CE2 151CZ 4.851   32CE2 36NH1 4.909   32CE3 33N 3.532   32CE3 33CA 3.828   32CE3 33CG1 4.157   32CE3 36CB 4.522   32CE3 155CD1 4.542   32CE3 33CB 4.649   32CE3 151CD 4.657

[0026]   32CE3 36CD 4.719   32CE3 151NE 4.929   32CD1 36NE 4.848   32NE1 36NE 3.919   32NE1 36CZ 4.139   32NE1 36CD 4.346   32NE1 36NH1 4.404   32NE1 36NH2 4.693   32CZ2 36CD 3.146   32CZ2 36NE 3.563   32CZ2 36CZ 3.945   32CZ2 36NH2 3.954   32CZ2 36CG 4.276   32CZ2 151CZ 4.401   32CZ2 151NE 4.403   32CZ2 151NH1 4.452   32CZ2 36CB 4.464   32CZ2 36NH1 4.873   32CZ2 151NH2 4.924   32CZ2 151CD 4.993   32CZ3 155CD1 3.624   32CZ3 151CD 4.106   32CZ3 36CD 4.225   32CZ3 151NE 4.250   32CZ3 151CG 4.430   32CZ3 36CB 4.488   32CZ3 33CA 4.545   32CZ3 33N 4.616

[0027]   32CZ3 36CG 4.653   32CZ3 33CG1 4.754   32CZ3 151CZ 4.802   32CH2 36CD 3.427   32CH2 151NE 3.956   32CH2 36CG 4.238   32CH2 36NE 4.297   32CH2 151CD 4.299   32CH2 151CZ 4.365   32CH2 155CD1 4.378   32CH2 36CB 4.459   32CH2 151NH1 4.669   32CH2 151CG 4.722   32CH2 36NH2 4.800   32CH2 36CZ 4.890   32C 33CA 2.430   32C 33C 3.009   32C 33O 3.730   32C 33CB 3.737   32C 36N 4.094   32C 33CG1 4.149   32C 36CB 4.453   32C 36CA 4.929   32C 33CG2 4.950   32O 33N 2.249   32O 33CA 2.757   32O 33C 2.945   32O 36N 2.962

[0028]   32O 33O 3.261   32O 36CB 3.270   32O 36CA 3.712   32O 33CB 4.267   32O 36CG 4.657   32O 37N 4.735   32O 36C 4.758   32O 33CG1 4.844   33N 36N 4.835   33CA 37OG1 4.386   33CA 36N 4.658   33CA 36CB 4.957   33CA 37N 4.991   33CA 37CG2 4.994   33CB 37OG1 4.651   33CG2 37OG1 3.631   33CG2 155CB 4.276   33CG2 155CD1 4.585   33CG2 155O 4.633   33CG2 37CG2 4.695   33CG2 155CG2 4.767   33CG2 37CB 4.789   33CG2 155CG1 4.874   33CG1 155CG2 4.351   33CG1 155CB 4.696   33CG1 155CD1 4.793   33CD1 155CG2 4.145   33CD1 155CB 4.658

[0029]   33C 37OG1 3.580   33C 36N 3.854   33C 37N 4.042   33C 37CB 4.576   33C 36CA 4.656   33C 37CG2 4.688   33C 36CB 4.779   33C 37CA 4.826   33C 36C 4.863   33O 37OG1 2.646   33O 37N 2.851   33O 36N 3.274   33O 37CB 3.467   33O 37CA 3.608   33O 37CG2 3.684   33O 36C 3.777   33O 36CA 3.840   33O 36CB 4.138   33O 37C 4.148   33O 36O 4.926   36N 37N 2.838   36N 37CA 4.277   36N 37C 4.949   36CA 37N 2.434   36CA 37CA 3.811   36CA 37CG2 4.500   36CA 37CB 4.704   36CA 37C 4.796

[0030]   36CB 37N 3.318   36CB 37CG2 4.430   36CB 37CA 4.592   36CG 37N 3.982   36CG 37CG2 4.535   36CG 37CA 4.970   36C 37CA 2.432   36C 37CG2 3.497   36C 37CB 3.498   36C 37C 3.538   36C 37OG1 4.176   36C 37O 4.442   36C 249CD 4.916   36C 249CG 4.954   36O 37N 2.243   36O 37CA 2.766   36O 37CG2 3.844   36O 37C 3.859   36O 249CD 3.882   36O 37CB 3.898   36O 249CG 4.005   36O 37O 4.477   36O 247CB 4.749   36O 247OD1 4.807   36O 37OG1 4.881   37CA 247CB 4.321   37CA 247CA 4.867   37CB 156CG2 4.663

[0031]   37CB 247CB 4.782   37CG2 155CD1 3.854   37CG2 156CG2 3.941   37CG2 155CG1 4.422   37CG2 156CB 4.991   37C 247CB 4.542   37C 247CA 4.609   37C 247C 4.810   37O 247CA 3.598   37O 247C 3.729   37O 247CB 3.853   37O 247N 4.926   37O 247O 4.938  112N 276OG 3.686  112N 305CA 3.963  112N 306N 4.333  112N 305C 4.677  112N 304O 4.709  112N 276CB 4.828  112N 305N 4.952  112N 276CA 4.966  112CA 276OG 3.624  112CA 276C 4.051  112CA 304O 4.061  112CA 276CA 4.136  112CA 305CA 4.225  112CA 276O 4.408  112CA 244NE2 4.434

[0032]  112CA 276CB 4.443  112CA 244CE1 4.710  112CA 304C 4.800  112CA 244CD2 4.813  112CA 305N 4.911  112CB 304O 3.148  112CB 244CE1 3.594  112CB 244NE2 3.694  112CB 305CA 3.781  112CB 304C 4.127  112CB 244ND1 4.129  112CB 276OG 4.216  112CB 276CA 4.217  112CB 244CD2 4.306  112CB 305N 4.436  112CB 276C 4.515  112CB 244CG 4.571  112CB 276CB 4.716  112CB 276O 4.728  112CB 306N 4.945  112CB 305C 4.962  112CB 274OD1 4.977  112SG 276OG 3.687  112SG 276CA 3.809  112SG 244CE1 3.853  112SG 276CB 3.982  112SG 244NE2 4.070  112SG 274OD1 4.127

[0033]  112SG 274ND2 4.259  112SG 304O 4.414  112SG 157CE2 4.485  112SG 274CG 4.632  112SG 276N 4.659  112SG 305CA 4.685  112SG 276C 4.686  112SG 244ND1 4.776  112SG 142CD1 4.820  112C 304O 3.861  112C 305CA 4.386  112C 304C 4.415  112C 276C 4.524  112C 303CB 4.545  112C 276O 4.551  112C 244CD2 4.605  112C 305N 4.661  112C 244NE2 4.671  112C 276OG 4.831  112C 244CG 4.958  112O 244CD2 3.728  112O 276O 3.809  112O 303CB 3.827  112O 244NE2 4.073  112O 276C 4.079  112O 244CG 4.177  112O 304O 4.251  112O 244CE1 4.632

[0034]  112O 244ND1 4.683  112O 276CA 4.831  112O 244CB 4.890  112O 304C 4.905  112O 304N 4.955  112O 303CA 4.975  142CA 157CB 4.754  142CA 205CD1 4.956  142CB 157CD2 3.797  142CB 157CG 4.058  142CB 157CB 4.165  142CB 205CD1 4.170  142CB 157CE2 4.469  142CB 276CB 4.757  142CB 157CD1 4.905  142CG 276CB 3.788  142CG 276OG 4.071  142CG 205CD1 4.377  142CG 157CD2 4.414  142CG 157CE2 4.706  142CD1 276CB 3.608  142CD1 276OG 3.655  142CD1 205CD1 3.719  142CD1 157CE2 3.740  142CD1 157CD2 3.885  142CD1 487CZ 4.116  142CD1 487CE1 4.134  142CD1 157CZ 4.454

[0035]  142CD1 157CG 4.706  142CD1 276CA 4.885  142CD2 487CE1 4.604  142CD2 205CD1 4.620  142CD2 276OG 4.759  142CD2 276CB 4.818  142C 157CB 4.133  142C 157CG 4.691  142O 157CB 4.323  142O 205CD1 4.386  142O 157CG 4.706  151N 152N 2.952  151N 152CA 4.351  151CA 152N 2.436  151CA 152CA 3.810  151CA 152C 4.558  151CA 155CG2 4.685  151CB 152N 3.099  151CB 152CA 4.414  151CG 152N 3.627  151CG 155CG2 4.303  151CG 155CD1 4.606  151CG 152CA 4.623  151CD 152N 4.899  151C 152CA 2.431  151C 152C 3.097  151C 152O 4.095  151C 155CG1 4.343

[0036]  151C 155CG2 4.371  151C 155N 4.563  151C 155CD1 4.597  151C 155CB 4.899  151O 152N 2.258  151O 152CA 2.779  151O 152C 2.947  151O 155CG1 3.212  151O 155CG2 3.411  151O 155N 3.421  151O 152O 3.701  151O 155CD1 3.721  151O 155CB 3.737  151O 155CA 4.176  152CA 155CG1 4.815  152CA 155CD1 4.922  152C 207CE 4.094  152C 155CG1 4.510  152C 156CG2 4.843  152C 155N 4.860  152O 207CE 3.274  152O 156CG2 3.855  152O 155CG1 4.323  152O 156CG1 4.363  152O 156CB 4.727  152O 155CD1 4.807  152O 156N 4.911  155N 156N 2.685

[0037]  155N 156CA 4.127  155N 156CG2 4.821  155N 157N 4.835  155N 156C 4.966  155CA 156N 2.427  155CA 156CA 3.765  155CA 156CG2 4.562  155CA 156C 4.769  155CA 156CB 4.784  155CA 157N 4.991  155CB 156N 3.352  155CB 156CA 4.554  155CB 156CG2 4.561  155CG2 156N 4.705  155CG1 156N 3.270  155CG1 156CG2 3.509  155CG1 156CA 4.310  155CG1 156CB 4.510  155CD1 156CG2 4.165  155CD1 156N 4.638  155C 156CA 2.383  155C 156C 3.439  155C 156CB 3.567  155C 156CG2 3.643  155C 157N 3.931  155C 156O 4.261  155C 156CG1 4.558  155O 156N 2.227

[0038]  155O 156CA 2.675  155O 156C 3.604  155O 156CB 3.915  155O 156CG2 4.089  155O 156O 4.103  155O 157N 4.363  156N 157N 2.981  156N 157CA 4.422  156CA 157N 2.466  156CA 157CA 3.825  156CA 157C 4.700  156CA 157O 4.854  156CA 157CB 4.870  156CA 157CD1 4.959  156CA 274N 4.980  156CB 157N 3.377  156CB 157CA 4.624  156CB 246O 4.688  156CB 274CB 4.791  156CB 157CD1 4.859  156CG2 157N 4.653  156CG1 157N 3.213  156CG1 157CD1 3.583  156CG1 157CE1 3.655  156CG1 157CG 4.050  156CG1 157CZ 4.179  156CG1 157CA 4.262  156CG1 157CD2 4.517

[0039]  156CG1 274CB 4.523  156CG1 157CE2 4.578  156CG1 157CB 4.670  156CG1 207CE 4.697  156CG1 274ND2 4.790  156CG1 246O 4.975  156CD1 274ND2 3.308  156CD1 274CB 3.331  156CD1 157CZ 3.561  156CD1 157CE1 3.563  156CD1 157N 3.712  156CD1 157CE2 3.715  156CD1 157CD1 3.722  156CD1 274CG 3.772  156CD1 157CD2 3.864  156CD1 157CG 3.875  156CD1 246O 4.111  156CD1 157CA 4.343  156CD1 274CA 4.694  156CD1 157CB 4.712  156CD1 274N 4.913  156CD1 274OD1 4.936  156C 157N 1.329  156C 157CA 2.420  156C 157C 3.305  156C 157CB 3.660  156C 157O 3.694  156C 274N 3.900

[0040]  156C 157CG 4.021  156C 274CB 4.195  156C 157CD1 4.329  156C 274O 4.565  156C 274CA 4.613  156C 157CD2 4.668  156O 157N 2.229  156O 157CA 2.717  156O 274N 2.729  156O 157C 3.324  156O 274CB 3.467  156O 274CA 3.614  156O 157O 3.892  156O 274O 3.950  156O 157CB 4.129  156O 274C 4.220  156O 157CG 4.518  156O 274CG 4.862  156O 157CD2 4.863  157N 274O 4.374  157N 274N 4.566  157N 274CB 4.672  157CA 274O 3.295  157CA 274N 4.244  157CA 274C 4.279  157CA 274CB 4.444  157CA 274CA 4.594  157CB 274O 3.745

[0041]  157CB 274C 4.921  157CG 274O 3.919  157CG 205CD1 4.661  157CG 274CB 4.767  157CG 274CG 4.946  157CD1 205CD1 4.256  157CD1 205CD2 4.394  157CD1 205CG 4.976  157CD2 274O 3.280  157CD2 274CG 3.915  157CD2 274CB 4.085  157CD2 274ND2 4.099  157CD2 274OD1 4.220  157CD2 274C 4.280  157CD2 205CD1 4.766  157CD2 274CA 4.819  157CE1 205CD2 3.643  157CE1 205CD1 3.963  157CE1 205CG 4.458  157CE1 207CE 4.729  157CE1 207SD 4.791  157CE2 274ND2 3.503  157CE2 274CG 3.728  157CE2 274OD1 4.078  157CE2 274O 4.205  157CE2 274CB 4.299  157CE2 205CD1 4.515  157CE2 274C 4.976

[0042]  157CZ 274ND2 4.083  157CZ 205CD1 4.113  157CZ 205CD2 4.153  157CZ 274CG 4.640  157CZ 205CG 4.775  157CZ 207SD 4.820  157C 274O 3.627  157C 274N 4.446  157C 274C 4.527  157O 274O 4.850  189N 207CA 4.847  189CA 207CA 4.417  189CA 207N 4.864  189CA 207CB 4.928  189CA 487CE2 4.997  189CB 306CA 4.135  189CB 212CG1 4.600  189CB 487CE2 4.841  189CB 306N 4.926  189CB 487CD2 4.995  189CG 306CA 3.750  189CG 487CE2 3.924  189CG 306N 4.212  189CG 487CD2 4.347  189CG 487CZ 4.911  189CG 207CG 4.991  189CD1 207CB 3.783  189CD1 207CG 3.907

[0043]  189CD1 207SD 4.051  189CD1 487CE2 4.116  189CD1 207CA 4.314  189CD1 205CD2 4.490  189CD1 487CZ 4.872  189CD1 207N 4.916  189CD1 487CD2 4.942  189CD2 306CA 3.467  189CD2 306N 3.480  189CD2 305C 4.060  189CD2 305O 4.557  189CD2 305CA 4.739  189CD2 212CG2 4.797  189CD2 212CG1 4.883  189CD2 306C 4.903  189CD2 487CE2 4.911  189CD2 212CB 4.963  189C 487CD2 4.060  189C 487CE2 4.144  189C 487O 4.648  189C 306CA 4.974  189O 487CD2 3.681  189O 487O 4.066  189O 487CE2 4.173  189O 487C 4.215  189O 306CA 4.247  189O 306C 4.621  189O 487CG 4.813

[0044]  205CG 487CZ 4.299  205CG 487CE2 4.667  205CD1 487CZ 4.050  205CD1 487CE2 4.824  205CD1 487CE1 4.931  205CD2 207CB 4.532  205CD2 207N 4.789  205C 207N 4.445  205O 207N 4.564  207CA 209N 4.326  207CB 209N 4.314  207CB 209CA 4.966  207CG 209N 3.475  207CG 209CA 3.821  207CG 212CG1 4.074  207CG 212CG2 4.903  207SD 209CA 4.461  207SD 209N 4.640  207SD 212CG2 4.851  207SD 212CG1 4.933  207CE 209CA 4.469  207CE 209N 4.711  207C 209N 3.159  207C 209CA 4.396  207O 209N 3.120  207O 209CA 4.341  209N 210N 3.152  209N 212CG1 4.281

[0045]  209N 210CA 4.540  209N 212N 4.914  209CA 210N 2.421  209CA 210CA 3.796  209CA 212CG1 4.372  209CA 210C 4.462  209CA 212N 4.662  209CA 210CB 4.826  209CA 212CG2 4.959  209CA 210CG 4.975  209C 210CA 2.434  209C 210C 3.026  209C 210CB 3.693  209C 212N 3.800  209C 210O 3.920  209C 210CG 4.126  209C 213N 4.177  209C 210OD1 4.376  209C 212CG1 4.433  209C 213CB 4.625  209C 212CA 4.667  209C 210ND2 4.747  209C 212CG2 4.782  209C 212CB 4.818  209C 212C 4.948  209O 210N 2.245  209O 210CA 2.777  209O 210C 2.905

[0046]  209O 213N 2.972  209O 212N 3.043  209O 210O 3.487  209O 213CB 3.674  209O 212CA 3.685  209O 212C 3.773  209O 212CG2 3.827  209O 213CA 3.906  209O 212CG1 3.916  209O 212CB 3.947  209O 210CB 4.249  209O 210CG 4.878  209O 212O 4.958  209O 210OD1 4.993  210N 212N 4.398  210N 213N 4.866  210N 213CB 4.979  210CA 213CB 4.405  210CA 212N 4.438  210CA 213N 4.596  210C 213N 3.886  210C 213CB 4.239  210C 213CA 4.591  210C 212CA 4.624  210C 212C 4.679  210O 213N 3.501  210O 213CB 3.633  210O 212N 3.644

[0047]  210O 213CA 3.933  210O 213C 4.126  210O 212C 4.352  210O 212CA 4.631  210O 213CG 4.674  210O 213CD1 4.727  212N 213N 2.784  212N 213CA 4.205  212N 213C 4.773  212N 213CB 4.895  212CA 213N 2.468  212CA 213CA 3.845  212CA 213C 4.420  212CA 216N 4.888  212CA 213CB 4.891  212CB 213N 3.397  212CB 213CA 4.698  212CB 304CE1 4.881  212CG1 213N 4.408  212CG2 213N 3.253  212CG2 213CA 4.264  212CG2 304CE1 4.815  212C 213N 1.335  212C 213CA 2.440  212C 213C 2.994  212C 213CB 3.710  212C 213O 3.755  212C 216N 3.855

[0048]  212C 216CB 4.183  212C 216CA 4.658  212C 213CG 4.966  212O 213N 2.245  212O 216N 2.670  212O 213CA 2.759  212O 213C 2.887  212O 216CB 3.134  212O 213O 3.255  212O 216CA 3.457  212O 213CB 4.240  212O 304CE1 4.399  212O 216C 4.660  212O 304CZ 4.701  213N 216N 4.607  213N 216CB 4.734  213CA 250CD1 4.134  213CA 216CB 4.363  213CA 216N 4.434  213CA 250CG1 4.898  213CA 216CA 4.958  213CB 250CD1 4.585  213CG 250CG1 4.288  213CG 250CD1 4.298  213CG 249NH2 4.361  213CD1 249NH2 4.189  213CD1 250CG1 4.969  213CD1 249CZ 4.976

[0049]  213CD2 250CG1 3.388  213CD2 250CD1 3.646  213CD2 247ND2 4.070  213CD2 247OD1 4.172  213CD2 249NH2 4.297  213CD2 249CB 4.423  213CD2 247CG 4.542  213CD2 250CB 4.579  213CD2 250N 4.650  213CD2 249CD 4.689  213CD2 250CA 4.924  213CE1 249NH2 3.969  213CE1 249CZ 4.403  213CE1 249NH1 4.789  213CE1 250CG1 4.914  213CE1 249NE 4.963  213CE2 250CG1 3.302  213CE2 249CB 3.341  213CE2 250N 3.738  213CE2 213CB 3.785  213CE2 247OD1 3.897  213CE2 249C 4.015  213CE2 249CD 4.057  213CE2 249NH2 4.080  213CE2 250CD1 4.089  213CE2 250CA 4.157  213CE2 250CB 4.248  213CE2 249CG 4.314

[0050]  213CE2 249CA 4.346  213CE2 249NE 4.420  213CE2 249CZ 4.459  213CE2 247ND2 4.468  213CE2 249O 4.492  213CE2 247CG 4.604  213CZ 249CB 3.765  213CZ 249NH2 3.909  213CZ 249CZ 4.112  213CZ 250CG1 4.148  213CZ 249NE 4.295  213CZ 249C 4.377  213CZ 249CD 4.394  213CZ 250N 4.428  213CZ 249O 4.508  213CZ 249NH1 4.708  213CZ 249CG 4.729  213CZ 250CA 4.749  213CZ 249CA 4.754  213C 216N 3.701  213C 216CB 4.305  213C 216CA 4.432  213C 250CD1 4.614  213C 216C 4.795  213O 216N 3.163  213O 212O 3.255  213O 213CB 3.375  213O 213CG 3.382

[0051]  213O 213CD1 3.423  213O 213N 3.471  213O 216CA 3.662  213O 216CB 3.709  213O 216C 3.731  213O 250CD1 4.129  213O 250CG1 4.545  213O 216O 4.914  216N 304CZ 4.099  216N 304CE2 4.314  216N 304CE1 4.419  216N 304CD2 4.807  216N 304CD1 4.895  216CA 304CE2 3.847  216CA 304CD2 3.926  216CA 304CZ 3.934  216CA 304CG 4.094  216CA 304CE1 4.099  216CA 304CD1 4.169  216CA 250CD1 4.669  216CA 304CB 4.908  216CA 220N 4.975  216CA 220CD1 4.997  216CB 250CD1 3.531  216CB 304CD1 3.550  216CB 304CE1 3.746  216CB 304CG 3.756  216CB 304CZ 4.110

[0052]  216CB 304CD2 4.118  216CB 304CE2 4.288  216CB 304CB 4.383  216CB 250CG1 4.685  216CB 220CD1 4.706  216C 220N 4.091  216C 220CG 4.389  216C 220CD1 4.410  216C 220CB 4.425  216C 250CD1 4.646  216C 304CD2 4.738  216C 304CE2 4.889  216C 220CA 4.901  216C 304CG 4.985  216O 220N 2.973  216O 220CB 3.240  216O 220CG 3.312  216O 220CD1 3.600  216O 220CA 3.682  216O 304CD2 4.466  216O 220CD2 4.723  216O 220C 4.729  216O 304CG 4.812  216O 304CE2 4.867  220CG 304CB 3.954  220CG 304CD2 4.620  220CG 304CG 4.655  220CG 304N 4.839

[0053]  220CG 303C 4.910  220CG 304CA 4.914  220CG 303O 4.942  220CD1 250CG2 3.858  220CD1 304CB 3.918  220CD1 304N 4.769  220CD1 304CG 4.781  220CD1 304CA 4.980  220CD2 303O 3.794  220CD2 303C 3.874  220CD2 304CB 4.033  220CD2 303N 4.091  220CD2 304N 4.170  220CD2 303CA 4.361  220CD2 304CA 4.531  220CD2 304CD2 4.978  220CD2 304CG 4.997  244N 303CA 4.590  244N 303N 4.800  244N 303CB 4.918  244CA 246N 4.644  244CA 303CA 4.703  244CA 303CB 4.756  244CB 303CA 3.618  244CB 303CB 3.663  244CB 304N 4.380  244CB 303N 4.545  244CB 303C 4.581

[0054]  244CB 246N 4.821  244CG 303CB 4.261  244CG 246N 4.437  244CG 303CA 4.495  244CG 304O 4.536  244CG 276O 4.605  244CG 304N 4.694  244CG 274OD1 4.945  244CD2 276O 3.276  244CD2 276C 4.179  244CD2 274OD1 4.296  244CD2 276CA 4.491  244CD2 276N 4.705  244CD2 303CB 4.764  244ND1 246N 3.736  244ND1 246CB 3.939  244ND1 304O 4.002  244ND1 246CA 4.065  244ND1 274OD1 4.288  244ND1 274ND2 4.528  244ND1 274CG 4.656  244ND1 304N 4.729  244CE1 274OD1 3.036  244CE1 274ND2 3.525  244CE1 274CG 3.527  244CE1 246N 4.024  244CE1 246CA 4.116  244CE1 246CB 4.253

[0055]  244CE1 304O 4.409  244CE1 276O 4.453  244CE1 276CA 4.503  244CE1 276N 4.607  244CE1 274CB 4.806  244CE1 276C 4.903  244NE2 274OD1 2.997  244NE2 276O 3.189  244NE2 276CA 3.620  244NE2 276N 3.747  244NE2 276C 3.774  244NE2 274CG 3.873  244NE2 274ND2 4.248  244NE2 246N 4.811  244C 246N 3.311  244C 246CA 4.639  244O 246N 3.012  244O 246CA 4.288  244O 246CB 4.440  246N 247N 2.858  246N 247CA 4.225  246N 247O 4.519  246N 274ND2 4.635  246N 274CG 4.694  246N 247C 4.783  246N 274CB 4.908  246CA 247N 2.398  246CA 274ND2 3.762

[0056]  246CA 247CA 3.780  246CA 274CG 4.159  246CA 274CB 4.398  246CA 247CB 4.470  246CA 247ND2 4.518  246CA 247O 4.632  246CA 247C 4.704  246CA 274OD1 4.840  246CA 247CG 4.866  246CB 247N 3.017  246CB 247ND2 3.981  246CB 274ND2 4.268  246CB 247CA 4.360  246CB 247CG 4.666  246CB 247CB 4.733  246CB 247O 4.816  246CB 250CG2 4.830  246CB 250CD1 4.837  246CB 250CB 4.978  246CB 274CG 4.988  246C 247CA 2.445  246C 247CB 3.093  246C 247C 3.647  246C 247ND2 3.730  246C 247CG 3.789  246C 247O 3.922  246C 274ND2 4.440  246C 274CB 4.631

[0057]  246C 247OD1 4.815  246C 274CG 4.829  246O 247N 2.265  246O 246CA 2.400  246O 247CA 2.826  246O 247CB 3.054  246O 247ND2 3.937  246O 247CG 3.998  246O 274ND2 4.116  246O 274CB 4.132  246O 247C 4.279  246O 274CG 4.548  246O 247O 4.812  247CA 249N 4.491  247CB 249N 4.494  247CG 250N 3.957  247CG 249N 4.038  247CG 250CB 4.274  247CG 249CB 4.318  247CG 250CG1 4.508  247CG 249CA 4.619  247CG 249CG 4.681  247CG 250CD1 4.751  247CG 250CA 4.762  247CG 249C 4.818  247OD1 249N 3.007  247OD1 250N 3.066  247OD1 249CB 3.116

[0058]  247OD1 249CA 3.431  247OD1 249CG 3.604  247OD1 249C 3.735  247OD1 250CB 4.015  247OD1 250CA 4.121  247OD1 249CD 4.172  247OD1 250CG1 4.267  247OD1 250CD1 4.870  247OD1 249O 4.930  247ND2 250CD1 3.820  247ND2 250CG1 3.953  247ND2 250CB 4.004  247ND2 250N 4.402  247ND2 250CA 4.869  247C 249N 3.305  247C 250N 4.120  247C 249CA 4.545  247C 249C 4.779  247C 250CB 4.900  247C 250CA 4.940  247O 249N 3.360  247O 250N 3.406  247O 250CB 3.950  247O 250CA 3.970  247O 250C 4.123  247O 249C 4.217  247O 249CA 4.373  247O 250CG2 4.591

[0059]  249N 250N 2.817  249N 250CA 4.203  249N 250C 4.666  249CA 250N 2.413  249CA 250CA 3.779  249CA 250C 4.413  249CA 250CB 4.866  249CB 250N 3.035  249CB 250CA 4.353  249CG 250N 4.416  249C 250CA 2.410  249C 250C 3.035  249C 250CB 3.720  249C 250O 3.774  249C 250CG1 4.278  249C 250CG2 4.919  249O 250N 2.240  249O 250CA 2.733  249O 250C 3.038  249O 250O 3.374  249O 250CB 4.232  249O 250CG1 4.716  274OD1 276CB 4.452  274OD1 276C 4.632  274OD1 276O 4.648  274ND2 276N 4.935  274C 276N 3.322  274C 276CA 4.688

[0060]  274O 276N 3.552  274O 276CA 4.809  303N 304CA 4.862  303CA 304N 2.430  303CA 304CA 3.818  303CA 304C 4.661  303CA 304O 4.679  303CA 304CB 4.684  303CB 304N 3.222  303CB 304CA 4.521  303CB 304O 4.818  303CB 304C 4.963  303C 304CA 2.452  303C 304CB 3.442  303C 304C 3.504  303C 304O 3.860  303C 305N 4.467  303C 304CG 4.748  303O 304N 2.247  303O 304CA 2.806  303O 304CB 3.762  303O 304C 3.989  303O 304O 4.630  303O 305N 4.674  303O 304CG 4.827  304N 305N 3.547  304N 305CA 4.719  304CA 305N 2.450

[0061]  304CA 305CA 3.795  304CA 305C 4.958  304CB 305N 3.325  304CG 305N 3.321  304CG 305CA 4.469  304CG 305O 4.729  304CD1 305N 3.304  304CD1 305CA 4.052  304CD1 305O 4.145  304CD1 305C 4.383  304CD2 305N 4.139  304CE1 305O 3.966  304CE1 305N 4.100  304CE1 305C 4.483  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

[0062]  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

Table III provides the atomic coordinates at the active site of the acetyl-CoA complex structure. Solvent molecules have been removed for clarity, but can be seen in FIG. Residue 487 is Phe87 derived from another monomer. The CAC residue is an acetylated cysteine and the COA is the bound CoA molecule.

[0064] Table III atoms residues 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 19.970 31. 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 32. 4.325 28.378 1.00 17.69 22 N ILE 33 25.53 6 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 2 1.00 16.21 27 CD1 ILE 33 28.930 4.480 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 35.315 355 26.63 38 NH2 ARG 36 24.330 23.355.355. 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 25.925 1.00 17.72 45 C G2 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. 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

[0065]  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

[0066]  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

[0067] 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

[0068] 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

[0069] 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

[0070] 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

[0071] 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

[0072] 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

[0073] 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 PO10 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

Variants and Derivatives The present invention further provides homologues, co-complexes and variants of the E. coli FabH crystal structure of the present invention. The term "homologue" means a protein having at least 30% amino acid sequence identity with E. coli FabH or any functional domain thereof. The term “co-complex” means FabH or a variant or homologue of FabH covalently or non-covalently bound to a chemical entity or compound. The term "variant" refers to a FabH polypeptide, ie, a wild-type FabH, characterized by substituting at least one amino acid from the wild-type FabH sequence.
Refers to a polypeptide displaying a biological activity of H activity. Such mutants can be prepared, for example, by expressing E. coli FabH cDNA whose coding sequence has been modified by oligonucleotide directed mutagenesis.

The E. coli FabH mutant also has a non-naturally occurring amino acid, CJNoren.
Et al., Science 244: 182-188 (1989), by site-specific incorporation into FabH protein. In this method, the codon encoding the amino acid of interest in the wild-type FabH enzyme is replaced with the "blank" nonsense codon, TAG, using oligonucleotide directed mutagenesis. The suppressor tRNA directed to this codon is then chemically aminoacylated in vitro with the desired non-naturally occurring amino acid. The aminoacyl-treated tRNA is then added to an in vitro translation system to yield a mutant FabH enzyme with incorporated site-specific unnatural amino acids.

Selenomethionine may be incorporated into wild-type or mutant FabH by expressing a cDNA encoding E. coli FabH in an auxotrophic mutant or a non-auxotrophic mutant E. coli strain ( WA Hendrickson et al., EMBO J., 9 (5
): 1665-1672 (1990)). In this method, the wild-type or mutated FabH cDNA can be expressed in a host organism that is depleted of natural methionine but in a growth medium rich in selenomethionine. The position of the Se atom can be determined by X-ray diffraction analysis of 3 or 4 different wavelengths.
In turn, this information is used to obtain face information used to construct the three-dimensional structure of the enzyme.

II. Method of Identifying Novel FabH Crystal Structure Inhibitors Another aspect of the present invention is to provide FabH enzyme inhibitors and inhibitors themselves characterized by the crystal structures and novel active sites described herein. Relates to a method for identifying. The novel E. coli FabH crystal structures or structures of E. coli FabH homologues of the present invention allow the identification of inhibitors of FabH activity. Such inhibitors may be competitive or non-competitive in binding to all or part of the FabH active site, whether it binds to FabH and it binds to another chemical entity. It inhibits FabH. One design method uses the Fa of the present invention using molecules composed of various different chemical entities.
To probe the bH crystals and determine the optimal site for interaction between the candidate inhibitor and the enzyme. For example, high resolution X-ray diffraction data collected from solvent saturated crystals can determine where each type of solvent molecule attaches. Then,
Small molecules that bind tightly to such sites can be designed and synthesized and tested for their FabH activity (J. Travis, Science 262: 1374 (1993).
)).

The present invention also enables the development of compounds that can be isomerized to short-lived reactive intermediates by the chemical reaction of FabH-binding substrates or other compounds that bind FabH. That is, it enables a time-dependent analysis of the structural changes of FabH during the interaction of FabH with other molecules. The reactive intermediate of FabH can also be inferred from the reaction product in a co-complex with FabH. Such information is based on known Fab
It is useful for designing improved analogues of H inhibitors, or for designing a series of novel inhibitors based on reactive intermediates of the enzyme and enzyme-inhibitor co-complexes. This provides a novel route for designing FabH inhibitors with both high specificity and stability properties. Another method enabled by the present invention is to computationally screen a small molecule database for chemical entities or compounds capable of fully or partially binding to FabH enzymes. In this screen, the suitability of such an entity or compound for the binding site may be determined from either shape complementarity or predicted interaction forces (EC Meng et al., J. Comp. C).
hem. 13: 505-524 (1992)).

Since FabH can crystallize in more than one crystalline form, the structural coordinates of FabH or portions thereof, as provided by the present invention, are useful in elucidating the structures of these other crystalline forms of FabH. Is. The coordinates can also be used to elucidate the structure of the FabH mutant co-complex or the crystalline forms of other proteins that have significant amino acid sequences homologous to the functional domains of FabH. One method that can be used for this purpose is the molecular replacement method. In this method, the unknown crystalline form is another crystalline form of FabH or a FabH variant,
FabH co-complex, FabH from various bacterial species, or F
Whether it is a crystal of another protein having a significant amino acid sequence homologous to any domain of abH, using the FabH structural coordinates of the invention as shown in FIG. 1 and Tables I-III, its unknown Crystal morphology can be measured. With this method, an accurate structural morphology for an unknown crystal would be obtained faster and more effectively than trying to determine such information from scratch.

Thus, the FabH structures presented herein bind to that structure, and in particular to the screening of known molecules that bind to the active site or substrate binding site, and / or to the structure using a computerized evaluation system. Enables the design of new molecules. For example, the sequence of FabH and the FabH structure (ie, FIG. 1 herein).
2 and Tables I-III, the computer modeling system in which the atomic coordinates, the bond distances between atoms in the active site region, and the like are input can be used. Thus, the instrument readable medium can also be encoded with the data indicating the coordinates of Figures 1-2 and Tables I-III. The computer can then obtain structural detail data for the site to which the test compound will bind, thereby determining the complementary detail structure of the test compound.

More specifically, the design of compounds that bind or inhibit FabH according to the present invention generally requires two factors to be considered. First, the compound must be physically and structurally capable of binding FabH. Non-covalent molecular interactions important in the binding of FabH to its substrate include hydrogen bonding, van der Waals forces and hydrophobic interactions. Second, the compound must be able to assume a conformation in which it can bind FabH. Although specific parts of the compound may not be directly involved in binding this FabH, these parts may still affect the overall conformation of the molecule. This may, in the end, have a significant impact on that possibility. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in the context of binding sites, eg, all or part of the active or substrate binding sites of FabH, or FabH. Includes spacing between the functional groups of compounds containing some chemical entities that interact directly.

The potential inhibitory or binding effects of a compound on FabH may be analyzed using computer modeling techniques before it is actually synthesized and tested. If the theoretical structure of a given compound does not suggest sufficient interaction and binding between the compound and FabH, synthesis and testing of the compound becomes unnecessary. However, if computer modeling indicates a strong interaction, then the molecule may be synthesized and tested for its ability to bind and inhibit FabH using an appropriate assay. In this way, the synthesis of ineffective compounds may be avoided. An inhibitor of FabH or other binding compound is computationally evaluated by a series of steps in which a chemical entity or fragment is screened and selected for its ability to bind to an individual binding pocket or other region of FabH, It can also be designed.

One of skill in the art will appreciate that FabH, and more specifically FabH, from among several methods.
One method can be used to screen a chemical entity or fragment for its ability to bind to the individual binding pockets of the active site or ancillary binding sites. This method is described, for example, in FabH of Figures 1-2 and Tables I-III.
One can start with a visual inspection of the active site on the computer screen, based on the coordinates. The selected fragment or chemical entity may then be oriented in various orientations or docked within the FabH binding pocket. For docking, use software such as Quanta and Sybyl, followed by CHA
It can be done using energy minimization and molecular mechanics in standard molecular mechanics force fields such as RMM and AMBER.

Specialized computer programs also aid in the steps for selecting fragments or chemical entities. These include the following programs: GRID [PJ Goodford, "A Computational Procedure for Determining Ene
rgetically Favorable Binding Sites on Biologically Important Macromolecu
les ", J. Med. Chem. 28: 849-857 (1985)]; GRID is UK, Oxford,
Available from Oxford University; MCSS [A.Miranker and M.Karplus, "Functionality Maps of Binding Si
tes: A Multiple Copy Simultaneous Search Method ", Proteins: Structure, F
unction and Genetics, 11: 29-34 (1991)]; MCSS is MA, Burlingt
on, available from Molecular Simulations; AUTODOCK [DS Goodsell and AJ Olsen, "Automated Docking of Subst
rates to Proteins by Simulated Annealing ", Proteins: Structure, Function
, and Genetics 8: 195-202 (1990)]; AUTODOCK is CA, La Jolla
, Scripps Research Institute; DOCK [IDKuntz et al., "A Geometric Approach to Macromolecule-Ligand I
nteractions ", J. Mol. Biol. 161: 269-288 (1982)]; DOCK is CA
, San Francisco, University of California.

Once the appropriate chemical entity or fragment is selected, it can be assembled into a single compound or inhibitor. The assembly may proceed by visually inspecting the interrelationship of the fragments in a three-dimensional image displayed on a computer screen in relation to the structural coordinates of the FabH. This is done by manual model building using software such as Quanta and Sybyl.

Useful programs to assist one of ordinary skill in the art in the context of individual chemical entities or fragments include: CAVEAT [PA Bartlett et al., "CAVEAT: A Program to Facilitate the Struc
ture-Derived Design of Biologically Active Molecules ", Molecular Recogni
tion in Chemical and Biological Problems, Special Pub., Royal Chem. Soc.
78, 182-196 (1989)]; CAVEAT is from CA, Berkeley, University.
available from of California; 3D database systems such as MACCS-3D (CA, San Leandro, MDL Information Systems); the technical scope is YCMartin, "3D Database Searching i
n Drug Design ", J. Med. Chem., 35: 2145-2154 (1992); 3. HOOK (MA, Burlington, Molecular Simulations).

FabH can be stepwise using one fragment or chemical entity described above at a time.
Instead of proceeding with the assembly of the inhibitor, the inhibitor or other FabH binding compound is generally or depleted using an empty active site or an active site which may optionally include a particular portion of the known inhibitor. May be designed. These methods are: 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 CA, San Diego, Biosym Technologies. 2. LEGEND [Y. Nishibata and A. Itai, Tetrahedron 47: 8985 (19
91)]; LEGEND is available from Molecular Simulations, Burlington, MA. 3. LeapFrog [available from MO, St. Louis, Tripos Associates].

Other molecular modeling methods may also be used in accordance with the present invention. For example, NCCo
hen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry"
, J. Med. Chem. 33: 883-894 (1990). Furthermore, M.
A. Navia and MA Murcko, "The Use of Structural Information in Drug Desi.
Gn ", Current Opinions in Structural Biology 2: 202-210 (1992).
)checking. For example, if the structure of the test compound is known, one model of the test compound may be overlaid with a model of the structure of the invention. Many methods and techniques are known in the art for performing this step, any of which may be used. For example, PSFarmer, Drug Design, Ariens, EJ, Volume 10, 119-1
Page 43 (Academic Press, New York, 1980); US Pat. No. 5,331,573; US Pat. No. 5,500,807; C. Verlinde, Curr. Biol. 2: 577-587.
(1994); and ID Kuntz, Science 257: 1078-1082 (19).
92). The model building techniques and computer evaluation systems described herein do not limit the scope of the invention.

Thus, these compounds can be used to quickly and easily test large numbers of compounds and avoid expensive and time consuming tests. Moreover, the need to actually synthesize many compounds is effectively eliminated. Once identified using modeling techniques, the FabH inhibitors may be tested for biological activity using standard techniques. For example, the constructs of the invention can be used in binding assays using conventional formats to screen for inhibitors. One particularly suitable assay format involves the enzyme-linked immunosorbent assay (ELISA). Other assay formats may be used;
These assay formats are not a limitation of the present invention.

In another aspect, the FabH structures of this invention allow the design or identification of synthetic compounds and / or other molecules characterized by the conformation of the FabH of this invention. The FabH of the present invention can be assayed using known computer systems.
The coordinates of the structure are obtained in instrument readable form, the test compound is designed and / or screened, and its conformation is superimposed on the FabH structure of the invention. The appropriate candidates identified above may then be screened for the desired FabH inhibitor biological activity, stability, etc. Once identified and screened for biological activity, these inhibitors can be used therapeutically or prophylactically to block FabH activity, ie bacterial replication.

The following examples illustrate various aspects of the present invention. These examples are not intended to limit the scope of the invention, which is limited to the claims set forth above. Example 1-FabH from Escherichia coli in Escherichia coli
Expression of Escherichia coli (Escherichia coli) as a host
The method for expressing FabH from E. coli is based on PCR amplification of the fabH gene and p
It was based on the introduction of suitable restriction sites allowing cloning of the FabH-containing DNA fragment in the ET29 expression vector. After PCR amplification, the insert of the resulting recombinant plasmid (hereinafter referred to as pET29c) was sequenced and the Taq
The absence of artifacts introduced by the polymerase reaction was confirmed. Expression was monitored by SDS-polyacrylamide gel analysis.

A. Bacterial strains, plasmids and media The Escherichia coli strain used was: MAXEfficiency DH10B Competent Cells Ge
notype: F- mcrA Δ (mrr-hsdRMS-mcrBC) φ80 dlacZ ΔM15 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).
All of these strains were commercial products. BL21 (DE3) competent cells for protein expression were purchased from Novagen. The protocol used to render it electroporation-functionalized was that provided by Invitorogen. The plasmid used was pET29 (Novagen). A detailed description of pET29 is shown in FIG. Briefly, pET29 is based on the T7 promoter drive system and allows selection of recombinant clones by kanamycin resistance.
E. coli expression vector.

LB medium per liter Baclo-tryptone 10 g Baclo-yeast extract 5 g NaCl 5 g For growth of the plasmid 0.1 mg / ml kanamycin was added to the medium.

B. DNA Manipulation Plasmid DNA was prepared by the rapid alkaline method (Sambrook et al., 198).
9). Transformation of E. coli cells was performed according to the protocol provided with DH10B or electroporation. The plasmid for sequencing was purified using the QIAGEN mini-prep kit (QIAGEN). Thermo Seque
DNA to supercoiled plasmid DNA by the dideoxy chain-termination method using the nce cycle sequencing kit (USA, ABI, BigDye, Applied Biosystems)
Sequencing was performed. Synthetic oligonucleotides (prepared in-house) were used as primers. Restriction enzymes and T4 DNA ligase were added to Gibco BRL (Life Technologie
s) and performed the experiment according to the instructions.

The FabH gene from E. coli cloned into the pET29 vector was amplified by PCR using the following primers; (5'-TATACATATGTATACGAAGATTATTGGT-3 '; SEQ ID NO: 2) and (5'-ATATGGATCCCTAGAAACGAACCAGCGCGG-3'. SEQ ID NO: 3). NdeI and BamHI restriction sites were added to the 5'and 3'of each primer, respectively.
It was incorporated at the end to facilitate ligation of the propagated DNA with the vector. Plasmid DNA (480 ng) was mixed with 200 μM deoxynucleotide triphosphate (dNTP), 0.20 mM oligonucleotide primer, manufacturer's buffer and 2.5 U pfu (Stratagene) PCR mix (10).
Amplification in 0 μl). The following cycling parameters were used: 94 ° C (4 minutes) 94 ° C (1 minute), 55 ° C (40 seconds), 72 ° C (1 minute); 30 cycles 72 ° C (2 minutes) 4 ° C. Polymerase chain reaction (PCR) is based on Gene Amp, PCR System 2400 [Perk
in Elmer Cetus Co.]. PCR-amplified DNA fragment with Qiaq
uick PCR Purification kit for Rapid Purification of DNA Fragments [Qiage
n].

C. Cloning of the E. coli fabH gene in the E. coli expression vector pET29 The cloning method is shown in FIG. PCR amplification of the fabH gene from E. coli was performed using primers AKK2 and AKK3, resulting in a DNA fragment of approximately 960 bp. This fragment was purified using the Qiaquick PCR Purification Kit protocol (Qiagen), digested with NdeI and BamHI, purified and ligated overnight to the NdeI and BamHI sites of the already digested vector pET29, resulting in the recombinant plasmid pET29c. Obtained. The ligation mixture was used to transform E. coli DH10B competent cells. pET29c
The construct was first purified from the transformants and confirmed by subjecting the plasmids to restriction analysis. Fa of pET29c, which is an essential step in amplification by Taq DNA polymerase
bH was sequenced and confirmed that no changes were introduced due to the low fidelity of this enzyme. Sequence analysis was performed using the T7 promoter and terminator primers. Sequencing of both strands revealed that no artifact was introduced during the amplification process.

D. Small-scale production of FabH from E. coli Plasmid pET29c and negative control pET29 (vector without insert)
Was used to transform the E. coli BL21 (DE3) host strain. BL21 (D
E3): A single clone of pET29c cells was grown overnight in the presence of 0.1 mg / ml kanamycin in 2 ml LB medium at 37 ° C. Then the cells
Diluted 100-fold with kanamycin in 0 ml LB. OD600 of culture is 0
． When the value of 5 was reached, FabH expression was induced by adding isopropyl-thio-galactoside (IPTG) at a final concentration of 0.5 mM. After this trigger,
2 ml samples were taken at different times (1 hour and 2 hours). Cells were harvested by microfuge for 3 minutes and pellet was 20 mM Tris-H.
Cl (pH8), washed with 1 mM PMSF, and resuspended in 100 μl SDS-PAGE gel loading buffer. The cells were disrupted by sonication (15 seconds). Then, the sample was boiled for 10 minutes, spin-treated once, and then 10 ml fractions were added to Laemmli (UK Laemmli, Nature 227, 680-685 (197).
It was analyzed by SDS-PAGE according to the method of 0)). 4-12% polyacrylamide gel (NOVEX) was stained with Coomassie blue. As shown in FIG.
Good expression levels were detected early after induction with IPTG. Evidence is for the presence of prominent bands (lanes 2, 4 and 6 in Figure 3) that were in good agreement with the molecular weight inferred from the primary sequence. FabH protein has a theoretical molecular weight of about 33514 Da.

Example 2-FabH Large Scale Growth and Purification Large-scale growth E. coli BL21 (DE3): 4 liter fermentation of pET29c with 100
Luria-Bertani medium (LB) containing kanamycin (μg / ml) was used. 8x
A second seed culture grown overnight in single strength LB medium containing 100 mg / ml kanamycin in a 500 ml flask was inoculated at 1% (v / v). Incubate the flask at 37 ° C, 250 rpm in a benchtop shaker.
It was stirred at. The OD at 600 nm was monitored, and when the absorbance was 0.5, isopropyl-thiogalactosidase was added to 0.5 mM to induce FabH expression, and 2 hours after the induction, the cells were centrifuged with a Sorval CSA rotor. And harvested.
A total of 20 g of cell paste was collected. The LB medium contains the following components per liter. This medium was supplied by our laboratory. Single strength baclo-tryptone 10 g Baclo-yeast extract 5 g Sodium chloride 5 g

B. Purification 1) Lysis E. coli FabH-overexpressing E. coli cells (12.5 g) obtained as described above were treated with 20 mM Tris (pH 7.9), 50 mM NaCl, and 1 mM.
mM EDTA, 1 mM DTT, 10% glycerol, 1 mM PMSF (
It was resuspended in 140 ml of buffer A). Lysozyme (Sigma Chemicals: chicken egg) was added to a final concentration of 1 mg / ml. The cells were incubated at 37 ° C for 20 minutes. The cells were then lysed by sonication (4 x 30 seconds) in an ice water bath. DNAse (Sigma; bovine pancreas type 1) was added with MgCl2 at 37 ° C.
Held for 5 minutes. Using a Beckman JA-14 rotor, the solution was 14000 g.
Centrifuge for 60 minutes in a Beckman JA-HS centrifuge.

2) Anion exchange All chromatographic procedures were carried out using a UV detector (Pharmacia, Monitor UV-
Performed on a Pharmacia chromatography system equipped with 1). UV (280 nm) was monitored during all runs. All operations were performed at 4 ° C. The supernatant from 1) was packed in a Pharmacia XK-26 column in 50 ml of Q-Se.
Loaded onto a pharose high performance (Pharmacia) column. The column was equilibrated with buffer A before loading. Then, the column was washed with buffer A (250
ml) at 4 ml / min and eluted with a linear gradient of buffer A to 1 M NaCl in buffer A over 4 min at 80 ml. The eluate was fractionated into 8 ml fractions using Pharmacia FRAC 200. The eluted fractions were assayed for FabH activity by measuring the uptake of [14C] acetyl-CoA into malonyl-ACP and for proteins by the Bradford method. SDS-PAGE (NOVEX, NuPAGE Bis-Tris 4-12
Active fractions were analyzed by reducing (% gradient gel). Active fractions were pooled together and dialyzed against buffer A.

3) Anion exchange chromatography The dialyzed material was loaded onto a Mono Q column (Pharmacia, 5/5) equilibrated with buffer A. The column was washed with 20 ml of equilibration buffer until the absorbance at 280 nm returned to baseline, then eluted with a linear gradient from equilibration buffer to buffer A at 0.5 ml / min for 90 minutes. Fractions were pooled together, pooled and assayed for FabH activity.

4) Hydroxyapatite / buffer exchange The eluted fractions were collected (1 ml fraction) and assayed for FabH activity and protein. The active fractions were pooled and the volume was reduced to half with buffer B (20 mM Tris-HCl (pH 7.4) to reduce the salt concentration in half.
, 50 mM NaCl, 1 mM DTT and 10% glycerol). Load the active eluate onto a hydroxyapatite column and add 0.5M
Elution with potassium phosphate (pH 7.4). 20 mM Tris pH 7.4, 50
The active enzyme was buffer exchanged using mM NaCl, 2 mM DTT. The product was more than 97% pure by SDS PAGE and the activity showed a yield of 60% in all steps from 1). N-terminal amino acid analysis confirmed the identity.

Example 3-Fermentation, Purification and Characterization of a Seleno-Methionine Derivative of Escherichia coli FabH Fermentation E. coli strain BL21 (DE3) was transformed with pET29c FabH to obtain soluble selmet-FabH for purification and crystallization experiments. 50 μl of seed culture expressing the FabH gene product was added to kanamycin (5
Luria broth (100 m) containing 0 μg / ml) and glucose (0.6%)
1). When the target density reached 2 OD, cells from the seed culture were isolated by centrifugation, containing 1 mM CaCl 2, 1 mM MgSO 4, kanamycin (50 μg / ml) and glucose (0.6% w / v), Resuspended in 100 ml M9 minimal medium. The resuspended pellet is then 900m
l of the same medium and the cells were grown at 37 ° C. to mid-log phase to an A600 of 1.5 at which time lysine, phenylalanine and threonine were each added at 100 mg / l.
, And selenomethionine, isoleucine and valine were added at 50 mg / l.
The cultures were shaken for 15 minutes and then induced with 0.5 mM isopropyl bD-thiogalactopyranoside (IPTG). The culture was grown for 13 hours and harvested by centrifugation (high speed). 5 ml before and during incubation
Was taken to monitor the expression of selenomethionine FabH. 5 g to 12 g of cell paste (wet weight) was recovered. In addition, one culture was prepared under the same conditions except that cells were grown in LB medium in order to compare the expression of the selenomethionine derivative with the expression of wtFabH.

B. Purification All lysis and purification steps were performed using degassed buffer on the anteroom or on ice. 4.5 g of E. coli cells overexpressing FabH were treated with 20 mM Tris,
50 mM sodium chloride, 10% glycerose, 0.2 mM PMSF, 2
It was resuspended in 50 ml of mM DTT (pH 7.9) (buffer A). Cells
10000ps using Microfluidizer (Microfluidics Corporation, MA)
Dissolved twice with i. Centrifuge cell debris at 35,000g for 30 minutes (Sorvall
RC-5B) and removed. 2.6 x 4 cm Sou equilibrated supernatant with buffer A
Rce Q column (Pharmacia). The column was loaded with 10 column volumes of buffer A
And washed with 10 column volumes of a gradient eluent of 0 to 1.0 M NaCl in buffer A. The eluted fraction was treated with 10% SDS-PAG under reducing conditions.
It analyzed by E. FabH was eluted with 0.2-0.25M NaCl. FabH
The containing fractions were pooled and applied to a 2.6 x 6 cm hydroxyapatite column (Bio-Rad, type I, 40 [mu]) equilibrated with buffer A. That column 3
Elution was with a linear gradient eluent from 0 column volume of buffer A to 400 mM potassium phosphate, 10% glycerol, 2 mM DTT. FabH was eluted with 80-180 mM potassium phosphate, 50 mM Tris, 200 mM NaCl,
1: 2 with 10% glycerose, 20 mM DTT (pH 7.5) (buffer B)
And was added to a 1.6 x 7.5 cm Blue Sepharose column (Pharcacia), which was equilibrated with Buffer B. The column was eluted with buffer B containing 1M NaCl. Next, the FabH fraction eluted with Blue Sepharose was added to 20 mM Tris, 5
2.6 × 60 equilibrated with 0 mM NaCl, 2 mM DTT (pH 7.4)
cm Superdex 200 size exclusion column (Pharmacia). 23 mg in total
FabH was collected and concentrated to 13 mg / ml using an Amicon 3 filter.

C. Characterization i) Mass Spectrometry Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-M
S) Data were obtained on a PerSeptive Biosystems Voyager RP laser desorption time-of-flight mass spectrometer. The analyte was added to 3,5-dimethoxy-4-hydroxycinnamic acid (0%) for a final concentration of 1-10 pmol / μl.
． 1: 10% / ml in 1% trifluoroacetic acid / acetonitrile (2: 1) 1:
A protein sample for analysis was prepared by diluting to 5. As an internal calibrator, bovine β-lactoglobulin A (Sigma) (MH +, 18364 Da) can be mentioned. Desorption /
Ionization was done using photon irradiation from a 337 nm pulsed nitrogen laser and 30 keV of driving energy. A spectrum was obtained by averaging about 100 laser scans. The estimated molecular weight of primordial FabH is 33516 Da. 3388 by MALDI-MS analysis of FabH protein constructs incorporating selenomethionyl
A mass of 9 Da was obtained. This value is in good agreement with the case where there is a shift of estimated +375 Da in the possible mass when the sulfur of eight methionine residues in the protein is replaced by the selenium side chain (theoretical value: 33891 Da).

Ii) N-terminal sequence analysis Hewlett-Pac to identify PTH online using HP1100 HPLC.
Sequencing was performed with a kard G1000A N-terminal protein sequencer. Samples were added directly to the biphasic sequencing cartridge and a standard 3.1 sequencing method cycle was used. N-terminal sequencing results showed negligible primitive methionine at the first residue. Instead, a unique PTH (eluting 1.6 minutes later than PTH-methionine
A phenylthiohydantoin) derivative was observed and did not co-elute with the natural PTH amino acid derivative. The increase in hydrophobicity is consistent with the direct detection of PTH-selenomethionyl amino acid derivatives.

D. Measurement of β-ketoacyl-ACP synthase III activity The enzyme catalyzes the condensation of acetyl-CoA and malonyl-ACP to form acetoacetyl-ACP. The reaction can be described in three different steps :(
a) the acyl group of acetyl-CoA is transferred to the active site cysteine to obtain an acyl-enzyme thioester; (b) the decarboxylation of malonyl-ACP to form a carbanion; and (c) the carbanion is acyl- Forming a carbon-carbon bond by nucleophilic attack on the carbonyl carbon atom of the enzyme thioester to give the acetoacetyl-ACP product.

To characterize the enzyme, or to identify specific inhibitors of its activity, 2
This reaction can be assayed in one of two ways: (1) Radiolabeled acetoacetyl-ACP formation is [3H] -acetyl-.
It can be specifically measured using CoA and malonyl-ACP. [3H
] -Acetyl-CoA substrate was soluble in 10% TCA, while obtained [3
[H] -acetoacetyl-ACP is not soluble. 100 mM sodium phosphate buffer (pH 7.0), 1 mM DTT, 34 μM acetyl-CoA, 0.
15 μM [3 H] -acetyl-CoA (specific activity 25 Ci / mmol) and 7
A reaction mixture containing μM malonyl-ACP was prepared and the inhibitor already added,
Alternatively, transfer to a microplate not added. The enzyme is added last to start the reaction and the plate is incubated at 37 ° C. The reaction is stopped by adding 10% TCA followed by 50 μg BSA as a carrier. The stopped reaction is filtered using a TomTec Harvester and washed twice on a Wallac GF / A filter with 10% TCA. The filter mats are dried at 60 ° C. and radioactivity quantified using a Wallac Betaplate scintillation cocktail and a Wallac Microbeta 1450 liquid scintillation counter. IC50s are obtained using GraFit 4.0 software and solved using the formula: v = Vmax / (1 + I / IC50).

(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) specifically reduces C3 carbonyl of acetoacetyl-ACP to produce β-hydroxybutyryl-ACP.
To form. This reduction requires converting NADPH to form NADP +, which can be monitored by following the optical density at a wavelength of 340 nm.

(3) FabD coupling is an assay option available in the absence of purified malonyl-ACP. In FabD (malonyl-CoA: ACP transacylase), malonic acid moves from malonyl-CoA to ACP, and malonyl-
It is involved in forming ACP. This activity is obtained by Fab using the above method (1).
It can be exploited by applying together with the new malonyl-ACP from the D reaction.

E. Ligand binding to FabH It is also possible to identify ligand interactions with FabH in experiments that do not rely on enzyme-catalyzed substrate turnover. This type of experiment can be done in many ways.

(1) Effect of Ligand Binding on Enzyme Intrinsic Fluorescence (eg of Tryptophan) The binding of either a natural ligand or an inhibitor causes a change in enzyme conformation that alters the fluorescence of the enzyme. Can bring. When using a stopped-flow fluorescence measuring device, this device can be used to limit the minimal rate constant that determines binding. Alternatively, the steady-state fluorometric titration method can yield a total dissociation constant for binding, as the method is accessed through enzyme inhibition experiments.

(2) Ligand Spectral Effects If the ligand itself has a fluorescent property or has a chromophore that overlaps with the enzyme tryptophan fluorescence, the ligand fluorescent property (eg intensity, lifetime or polarity).
Binding can be detected either through changes in or through fluorescence resonance energy transfer with the enzyme tryptophan. The ligand can be either an inhibitor or a variant of the natural ligand.

(3) Thermal Analysis of Enzyme: Ligand Complexes Calorimetric methods (eg, isothermal calorimetry, differential scan calorimetry) can be used to measure thermal changes indicative of ligand binding or FabH stability shifts. It is possible to detect and thus characterize its binding.

Example 4-Crystallization of E. coli wild type and selenomethionine mutants of FabH Crystallization All crystals were grown at room temperature using the sitting drop vapor diffusion method. The drop solution was always a 1: 1 mixture of protein sample and well solution.
In the case of the crystalline form of the wild-type protein, the well solution is 0.1 M HEPES buffer (pH
7.5) and 20% PEG8000. For crystal form 2 of the selenomethionine mutant protein complexed with acetyl-CoA, the well solution contained 0.05M Bis-Tris propane buffer (pH 7.0), 0.1M MgCl2 and 14% PEG6000. The crystals were grown overnight and were approximately 0.1 to 0.2 mm in size.

B. X-Ray Diffraction Characterization All crystals were frozen in a stream of liquid nitrogen before being characterized using synchrotron X-ray radiation. Diffraction data for apo crystal form 1 was collected to a resolution of 2.0Å. The data are 97.1% complete, 6-fold redundant and 7.7% merging R-factor. The crystal has a unit length of a = 63.1, b = 65.1.
And c = 166.5Å belong to the orthorhombic space group P212121. For the Se-Met protein complexed with acetyl-CoA, the data were collected at three different wavelengths: 0.9.
789, 0.9785 and 0.9414 Å. The three data sets had about 80% completeness, 6-fold redundancy, 8.5% merging R-factor, and 1.9Å resolution. Crystal form 2 has a = b = 72.4 and c =
It belongs to the tetragonal space group P41212 of 102.8Å.

C. Structural analysis The crystal structure of the Se-Met E. coli FabH mutant complexed with acetyl-CoA was collected at three different wavelengths and the program SOLVE (Terwil).
Linger & Berendzen, Acta Cryst. D55, 849-861 (1999)) MA
Using the D facing technique, the resolution up to 1.9Å was clarified. All eight S
e-Met was positioned by SOLVE. The advantage of the entire MAD facing diagram is 0.
The resolution was 6 to 1.9Å, and the overall Z score was high at 148. The electron density map obtained was of very high quality. The structure of the apo enzyme (crystal form 1) was elucidated by the molecular replacement method using the acetyl-CoA complex structure as a search model. This crystalline form had an asymmetric unit of FabH dimer and the R-factor of the solution was only 33%. A two-fold averaged map was then calculated and used for model building.

D. Model building and refinement The electron density for the acetyl-CoA complex was very sharp and a structural model for the entire FabH protein, bound acetyl groups and CoA, and 98 solvent molecules was assembled in the first round. An up-to-date model was obtained with standard structural refinement protocols and manual model building, which had an R-factor of -1 or 27%.
． It has a resolution of 9Å. A model of the apoFabH structure was also easily constructed,
Refining to an R-factor of 18.9% (Rfree of 24.4%) to a resolution of 2.0%. Both models have excellent external morphology,
There is no outlier in the manchandran) plot, indicating that the atomic coordinates are of high quality, including an estimation error of less than 0.3Å.

The present invention should not be limited to the embodiments described herein. Indeed, various modifications, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also within the scope of the claims. The patents, patent applications and publications cited herein are incorporated herein by reference in their source.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the atomic coordinates of the E. coli FabH dimer.

FIG. 2 shows atomic coordinates of E. coli FabH monomer complexed with acetyl-CoA.

FIG. 3 shows a projected view of a ribbon diagram of the E. coli FabH dimer. The two monomers are shown in light dark gray shading. Catalytic Cys 112 is shown with a dark ball-and-stick model.

FIG. 4 shows a ribbon diagram of the E. coli FabH monomer in which the catalytic residue Cys112 is shown in a dark ball-and-stick model. Label the N-good C-terminus.

FIG. 5 shows a stereogram with overlapping α-carbons between FabH and FabF structures. FabH is drawn with a thin black line and FabF is drawn with a thick gray line.

FIG. 6 shows a ribbon diagram of E. coli FabH monomer with acetylated Cys112 and CoA molecules in a ball-and-stick model. The orientation of this figure is the same as that of FIG.

FIG. 7 shows the E. coli FabH catalytic residues overlaid in comparison to the FabF residues. FabH is drawn as a thick gray line and FabF is drawn as a thin black line.
FabH residues are labeled with Cys112, His244 and Asn274, which correspond to Cys163, His303 and His340, respectively.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) (C12N 9/00 C12N 9/00 C12R 1:19) C12R 1:19 (C12Q 1/25 C12R 1:19 (71) Applicant SmithKline Beechham Public Limited Company SmithKline Beecham p. l. c. United Kingdom Tida Brew 89 GS, Middlesex, Brentford, Great West Road 980 (72) Inventor Cheryl Ann Jeanson United States 19010 Ladbroke Road, Bryn Ma, PA 200 (72) Inventor Kiu Shayan United States 19403 Lloyd Rain, Audubon, PA 2607 No. 2607 F-term (reference) 2G045 AA40 BA13 BB02 BB03 BB10 BB18 BB20 CB21 DA20 DA36 FA11 FB04 FB06 JA01 4B050 CC07 DD02 LL01 LL03 4B063 QA20 QQ40 5B075 UU

Claims (27)

[Claims] 1. A composition comprising crystalline form E. coli FabH. 2. The composition according to claim 1, wherein FabH is a dimer. 3. FabH comprises Cys112, His244 and Asn27.
4. An active site cavity formed by an amino acid comprising 4;
The composition as described. 4. The composition of claim 1, wherein FabH is E. coli FabH. 5. The composition of claim 3, wherein FabH is characterized by coordinates selected from the group consisting of the coordinates set forth in Figures 1-2 and Tables I, II and III. 6. An E. coli FabH crystal. 7. A selenomethionine mutant crystal of E. coli FabH. 8. An isolated and 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. As shown in FIG. 3, the molecule is a dimer, where each monomer is characterized by two similar domains with a core of 5 β-strands, each of which is 9. A molecule according to claim 8 which contains flanking helices, strands and loops. 10. The molecule of claim 8 wherein the molecule is a dimer characterized by the dimer interface of FIG. 11. The molecule of claim 10, which is E. coli FabH. 12. A peptide, peptidomimetic or synthetic molecule that interacts competitively or non-competitively with the active site of FabH of claim 1. 13. A method of identifying an inhibitory compound having the ability to bind to E. coli FabH and inhibit its enzymatic activity, the stereochemistry being defined by the coordinates of FIG. 1-2 and Tables I, II and III. The information specifying the conformation of the active site of the E. coli FabH molecule, including its conformation, is put into a suitable computer program; where the program displays its three-dimensional structure; Create a program; display and overlay a model of the test compound on the model of the active site; evaluate whether the test compound model spatially fits the active site; Fab characterized by site
A method for incorporation into a biological activity assay for H; determining whether the test compound inhibits enzymatic activity in the assay. 14. The FabH molecule is a dimer, as shown in FIG. 3, wherein each monomer is characterized by two similar domains with a core of 5 β-strands, 14. The method of claim 13, each containing flanking helices, strands and loops. 15. A method for identifying an inhibitory compound having the ability to bind to E. coli FabH and inhibit its enzymatic activity, the stereochemistry being defined by the coordinates of FIG. 1-2 and Tables I, II and III. The information specifying the conformation of the active site of the FabH molecule, including its conformation, is put into a suitable computer program; where the program displays its three-dimensional structure; Superimposing and displaying a model of the test compound on the model of the active site; assessing whether the test compound model spatially fits the active site; characterizing the test compound by the active site Incorporated into a biological activity assay for FabH; whether the test compound inhibits enzymatic activity in the assay. A method characterized by determining. 16. The FabH molecule is a dimer, as shown in FIG. 3, wherein each monomer is characterized by two similar domains with a core of five β-strands, 16. The method of claim 15, each containing flanking helices, strands and loops. 17. A peptide, peptidomimetic or synthetic molecule identified by the method of claim 13 or 15. 18. Elucidation of a crystal form, which comprises elucidating the crystal form of the FabH mutant, homologue or co-complex by molecular rearrangement using the structural coordinates of E. coli FabH crystal or a part thereof. Method. 19. E. coli F using the structural coordinates of E. coli FabH crystal
A method for designing a drug, which comprises a step of using a computer to evaluate a chemical entity for association with the active site and substrate binding site of abH. 20. The method according to claim 19, which comprises the step of using the structural coordinates of E. coli FabH to identify an intermediate in the chemical reaction between FabH and a compound that is a substrate or inhibitor of the enzyme. Design method. 21. The method of claim 20, wherein the entity is a competitive or non-competitive inhibitor of E. coli FabH. 22. Having similar amino acid identities and spatial arrangements with the amino acid identities and spatial arrangements of E. coli FabH listed in Tables I-III.
The method for designing a drug according to claim 19, wherein a structure of FabH homolog is used. 23. Having similar amino acid identities and spatial arrangements with the amino acid identities and spatial arrangements of E. coli FabH listed in Tables I-III.
The method for designing a drug according to claim 20, wherein a structure of FabH homolog is used. 24. Having similar amino acid identities and spatial arrangements to the E. coli FabH amino acid identities and spatial arrangements listed in Tables I-III.
The method for designing a drug according to claim 21, wherein the structure of FabH homolog is used. 25. The method of claim 19, wherein the structural coordinates include the coordinates of Figures 1-2 and Tables I, II and III. 26. The method of claim 20, wherein the structural coordinates include the coordinates of FIGS. 1-2 and Tables I, II and III. 27. The method of claim 21, wherein the structural coordinates include the coordinates of FIGS. 1-2 and Tables I, II and III.