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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
  • G16B15/30—Drug targeting using structural data; Docking or binding prediction
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
  • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

• the invention relates to the identification of a novel enzyme active site and methods enabling the design and selection of inhibitors of that active site.
• the pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes.
• Vertebrates possess a type I fatty acid synthase (FAS) in which all of the enzymatic activities are encoded on one multifunctional polypeptide, the mature protein being a homodimer.
• FAS fatty acid synthase
• ACP acyl carrier protein
• ACP is an integral part of the complex.
• ACP acyl carrier protein
• Mycobacteria are unique in that they possess both type I and II FASs.
• malonyl-ACP is synthesized from ACP and malonyl- CoA by FabD, malonyl CoA:ACP transacylase.
• malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively).
• the second step in the elongation cycle is ketoester reduction by NADPH-dependent ⁇ -ketoacyl-ACP reductase (FabG).
• FabH enzymes are of interest as potential targets for antibacterial agents.
• the present invention provides a novel FabH enzyme active site crystalline form.
• the present invention provides a novel FabH composition characterized by the catalytic residues Cysl 12, His244 and Asn274.
• the present invention provides a novel FabH composition characterized by the active site of 33 amino acid residues (including the catalytic residues).
• the invention provides a method for identifying inhibitors of the compositions described above which methods involve the steps of: providing the coordinates of the structure of the invention to a computerized modeling system; identifying compounds which will bind to the structure; and screening the compounds identified for FabH inhibitory bioactivity.
• the present invention provides an inhibitor of the catalytic activity of any composition bearing the catalytic domain described above.
• Another aspect of this invention includes machine readable media encoded with data representing the coordinates of the three-dimensional structure of the FabH crystal.
• Fig. 1 provides the atomic coordinates of the E. coli FabH di er.
• Fig. 2 provides the atomic coordinates of the E. coli FabH monomer in complex with acetyl-CoA.
• Fig. 3 provides a projection of the ribbon diagram of the E. coli FabH dimer. The two monomers are drawn with a light or dark gray shading. The catalytic Cysl 12 is shown in dark ball-and-stick model.
• Fig. 4 provides the ribbon diagram of the E. coli FabH monomer with the catalytic residue Cysl 12 is shown in dark ball-and-stick model. The N- and C-termini are labeled.
• Fig. 5 provides the stereoview of the oc-carbon superposition between the structures of FabH and FabF. FabH is drawn in a thin black line and FabF in a thick gray line.
• Fig. 6 provides the ribbon diagram of the E. coli FabH monomer with acetylated Cysl 12 and the CoA molecule in black ball-and-stick model. The orientation of the view is the same as that of Fig. 4.
• Fig. 7 provides the superposition of the E. coli FabH catalytic residues in comparison to those of FabF.
• FabH is drawn in thick gray lines and FabF in thin black lines.
• FabH residues are label Cysl 12, His244 and Asn274, which corresponds to Cys 163, His303 and His340, respectively.
• the present invention provides a novel E. coli FabH crystalline structure, a novel FabH active site, and methods of use of the crystalline form and active site to identify FabH inhibitor compounds (peptide, peptidomimetic or synthetic compositions) characterized by the ability to competitively inhibit binding to the active site of a FabH enzyme. Also provided herein is a novel FabH crystalline structure in complex with the substrate acetyl- CoA, and the identification of acetyl-CoA interacting residues in FabH.
• the present invention provides a novel FabH crystalline structure based on the E. coli FabH.
• the amino acid sequences of the FabH are provided in Table 1 as SEQ ID NO:l.
• the crystal structure is a tightly associated FabH dimer.
• Each monomer has two structural domains: the N-terminal domain (residues 1-170 of SEQ ID NO: l) and the C-terminal domain (residues 171-317 of SEQ ID NO: l).
• the two domains are similar in their overall fold: each contains a 5-stranded ⁇ -sheet sandwiched between ⁇ - helices and covered by other ⁇ -strands, ⁇ -helices and loops.
• the structural similarity between the two halves of the protein indicates that FabH is probably evolved from two genes of similar origin.
• the active site of FabH is at the center of the FabH monomer, formed at the junction of the N- and C-terminal domains. While the core architecture of the E. coli FabH bears some similarity to that of the FabF (Huang, et al, (1998), EMBO J. 17, 1183-1191), large differences exit in the atomic positions of the core ⁇ -strands, and the structures outside of the core ⁇ -strand are completely different. With amino acid sequence identity between FabH and FabF being below 20%, the large differences are well expected. Therefore, the crystalline structure ofE. coli FabH is novel.
• the E. coli FabH is a dimer, each monomer contains an active site.
• the dimer formation is essential for the FabH activity because the active site of a monomer is comprised of at least Phe87 of the other monomer in the dimer.
• the present invention provides both a crystalline monomer and dimer structure of E. coli FabH. Inhibitors that perturb or interact with this dimer interface are another target for the design and selection of anti-bacterial agents.
• the crystal structure of E. coli FabH has been resolved at 2.0 A (crystal form 1), and its selenomethionine mutant protein in complex with acetyl-CoA has been determined at 1.9 A (crystal form 2).
• the structure was determined using the methods of MAD phasing and molecular replacement, and refined to R-factors of 18.9% and 27%, respectively.
• FIG. 1 provides the atomic coordinates of the E. coli FabH dimer, which contains 634 amino acids.
• Figure 2 provides the atomic coordinates of the E. coli FabH monomer in complex with acetyl-CoA, which contains 317 amino acids.
• the FabH enzyme is characterized by an active site which preferably contains a binding site for the first substrate acetyl-CoA and the second substrate malonyl-ACP.
• the catalytic residues in FabH are Cysl 12, His244 and Asn274, compared to Cys 163, His303 and His340 in FabF.
• the difference in catalytic residues is not only limited to their amino acid identity (His340 to Asn274 change), but also their relative spatial arrangement. While FabH Cys 1 12 and Asn274 can be well superimposed onto FabF Cys 163 and His340, His244 of FabH occupies a very different position from that of His303 of FabF. This indicated the catalytic mechanisms of the two enzymes are very different.
• the crystal structure described herein was solved in the presence and absence of acetyl-CoA.
• the same acetyl-CoA binding cavity should bind malonyl-ACP as well because their active site binding regions are very similar and there is no apparent additional entrance to the active site.
• the FabH molecular surface in general negatively charged, a region just outside of the active site cavity is positively charge.
• This surface is mainly comprised of three ⁇ -helices (30-37, 209-231 and 248-258) and contains a number of positively charged amino acids (Arg36, Arg40, Lys214, His222, Arg235 Arg249, Lys256, Lys257). Since the acyl-carrier protein (ACP) is known to be very acidic or negatively charged, it is reasonable to assume this surface being the ACP binding surface.
• Table I provides the the atomic coordinates of the apo E. coli FabH structure in the active site (in crystal form 1). Solvent molecules are omitted here for clarity, but can be found in Fig. 1. Residue 487 is Phe87 from the other monomer. TABLE I
• Table II provides the distances between (D) atoms of the active site residues that are within 5.0 angstroms of one another as defined by Table I.
• Table III provides the the atomic coordinates of the acetyl-CoA complex structure in the active site. Solvent molecules are omitted here for clarity, but can be found in Fig. 2.
• Residue 487 is Phe87 from the other monomer.
• Residue CAC is acetylated cysteine, and COA is the bound CoA molecule.
• CD1 ILE 33 28. .930 4, .480 24. .013 1. .00 24, .09

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• 2000
  • 2000-06-07 EP EP00939638A patent/EP1183265A4/de not_active Withdrawn
  • 2000-06-07 WO PCT/US2000/015659 patent/WO2000075169A1/en not_active Application Discontinuation
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