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).

FIG. 1.

Roles of individual enzymes in a type II fatty acid synthase. Full enzyme names are provided in the text. Enzymes inhibited by triclosan, cerulenin, and TLM are indicated (wavy lines).

FIG. 2.

Structures of saFabH inhibitors tested in this study.

FIG. 3.

Multiple sequence alignment of saFabH and other bacterial homologues. The amino acid sequence of saFabH is compared with those of bFabH1, ecFabH, and mtFabH. Positions are numbered based on the sequence of ecFabH. The conserved triad (Cys 112, His 244, and Asn 274) at the enzyme active site is in bold face. The asterisks denote sequence identities, and the dots denote sequence similarities.

FIG. 4.

SDS-PAGE analysis of purified saFabH.

FIG. 5.

Molecular mass determination of native-form FabH using size exclusion chromatography. Log molecular mass versus elution volume/void volume ratio (Ve/Vo) is shown. The molecular mass standards used were β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa). Vo was determined using blue dextran (2,000 kDa). The saFabH mass was measured at approximately 67 kDa.

FIG. 6.

A pH profile for β-ketoacyl-ACP synthase activity of saFabH. Conditions were as described in Materials and Methods. Each datum point is the average of two determinations.

FIG. 7.

Kinetic analysis of saFabH for acetyl-CoA, butyryl-CoA, and MACP. The initial velocities of product formation were measured with purified saFabH and MACP in the presence of increasing concentrations of acetyl-CoA (A), butyryl-CoA (B), and isobutyryl-CoA (C). The initial velocities of product formation were measured with purified saFabH and butyryl-CoA in the presence of increasing concentrations of MACP (D). Each datum point is the average of three determinations.

FIG. 8.

Inhibition of saFabH by compounds HR12 (A) and HR19 (B). Inhibitors were tested using a TCA precipitation assay as described in Materials and Methods (n = 3). Inhibitor structures are shown in Fig. 2. conc., concentration.