title: Mechanism and inhibition of saFabI, the enoyl reductase from Staphylococcus aureus.
abstract: Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.
authors.affiliation: Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.forename: Todd J
authors.initials: TJ
authors.lastname: Sullivan
authors.forename: Jun-ichiro
authors.initials: J
authors.lastname: Sekiguchi
authors.forename: Teruo
authors.initials: T
authors.lastname: Kirikae
authors.forename: Iwao
authors.initials: I
authors.lastname: Ojima
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Stratton
authors.forename: Weimin
authors.initials: W
authors.lastname: Mao
authors.forename: Fernando L
authors.initials: FL
authors.lastname: Rock
authors.forename: M R K
authors.initials: MR
authors.lastname: Alley
authors.forename: Francis
authors.initials: F
authors.lastname: Johnson
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Walker
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tonge
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D010647:Phenyl Ethers / Q000494:pharmacology; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010647:Phenyl Ethers; C031538:phenyl ether; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)