title: Purification, characterization, and identification of novel inhibitors of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus.
abstract: Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent K(m) for malonyl-ACP was 1.76 +/- 0.40 microM, and the enzyme was active with acetyl-CoA (k(cat), 16.18 min(-1); K(m), 6.18 +/- 0.9 microM), butyryl-CoA (k(cat), 42.90 min(-1); K(m), 2.32 +/- 0.12 microM), and isobutyryl-CoA (k(cat), 98.0 min(-1); K(m), 0.32 +/- 0.04 microM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 microM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 +/- 0.10 microM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 +/- 0.08 microM).
authors.affiliation: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: He
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Reynolds
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016106:Methicillin Resistance; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009842:Oligopeptides; C444459:RWJ-3981; D013438:Sulfhydryl Compounds; D013876:Thiophenes; C418163:cysteine-N(methyl)valine-N(cyclohexyl)glycine-methionine-O-methyl-ester; C035068:thiolactomycin; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase