Crystal structure and substrate specificity of the β‐ketoacyl‐acyl carrier protein synthase III (FabH) from Staphylococcus aureus

Abstract

b-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 Å resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl-> hexanoyl-> butyryl-> isovaleryl-> > acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.Keywords: Staphylococcus aureus; FabH; X-ray crystallography; kinetics; substrate specificity Bacterial b-ketoacyl-ACP synthase (KAS) enzymes are important in the elongation steps of fatty acid biosynthesis Khandekar et al. 2003). KAS I (FabB) and KAS II (FabF) are involved in the condensation of malonyl-ACP with a growing acyl-ACP chain to form b-ketoacyl-ACP, which is a substrate for b-ketoacyl-ACP reductase (FabG). KAS III (FabH) catalyzes the initiation of fatty acid biosynthesis by condensing malonyl-ACP with acetyl-CoA. FabH from Gram-negative Escherichia coli has been studied extensively. It is encoded by the fabH gene, and is a homodimer with a monomeric molecular weight of 35 kDa (Han et al. 1998;Khandekar et al. 2003). It has been cloned, expressed, and purified by several groups, and has been extensively characterized both mechanistically and structurally (Qiu et al. 1999a;Davies et al. 2000;Khandekar et al. 2000). The E. coli FabH crystal structure has been solved in the presence and the absence of the substrate, acetyl-CoA (Qiu et al. 1999a(Qiu et al. , 2001Davies et al. 2000). In the crystal structure, the close approximation of Cys112 to CoA suggests it may play an important role in catalysis. Modeling based on a bound CoA molecule has identified His244 and Asn274 as additional residues that might be involved in catalysis.Staphylococcus aureus (S. aureus) is a Gram-positive human pathogen that causes diseases in humans, including skin infections, scalded-skin syndrome, and toxic

Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

“…8A). The FAS initiation activity of PA5174 is on par with E. coli FabH and with previously reported data (13,63).…”

“…8A). The FAS initiation activity of PA5174 is on par with E. coli FabH and with previously reported data (13,63).We next examined the reaction product of PA5174 using conformation-sensitive urea-PAGE with [2-14 C]malonyl-ACP (68). The putative ␤-acetoacetyl-ACP product of PA5714 migrates faster than the starting substrate [2-…”

Polyketide Synthase Gene Diversity within the Microbiome of the Sponge Arenosclera brasiliensis, Endemic to the Southern Atlantic Ocean

“…A total of 235 of these KS contigs encoded uninterrupted protein sequences of at least 150 amino acids; these results demonstrated that the quality of the data was high and sufficient for further phylogenetic analysis. These contigs also revealed the presence of conserved cysteine and histidine residues in the catalytic triad (CHH) of active KS domains from type I PKSs (38,39) as well as the primer-attaching conserved motifs DPQQR and HGTGT (40, 41) (see Fig. S2 in the supplemental material).…”

Synthesis, Molecular Modeling, and Quantitative Structure–activity Relationship Studies of Undec-10-Enehydrazide Derivatives as Antimicrobial Agents

“…The energy minimization was carried out by MM2 and finally by RHF/3-21G. Automated docking was used to determine the orientation of inhibitors bound in the active site of bacterial beta-ketoacyl-acyl carrier protein synthase III (FabH; pdb id:3IL7) [29,38]. An incremental construction algorithm method, implemented in the program FlexX embedded LeadIT, was employed.…”

“…Molecular docking studies were also carried out to understand the binding pattern and to support in vitro antimicrobial data of synthesized most active compound [27,28]. Automated docking technique was used to determine the orientation of inhibitors, bound in the active site of β-ketoacyl-acyl carrier protein synthase III (FabH; pdb id:3IL7) [29]. β-ketoacyl-acyl carrier protein synthase encoded by the fabH gene is reported to catalyze the first : 3076.56 (C-H str., alkenes), 2928.04 (C-H str., aliphatic), 1741.78 (C=O str.…”