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

In the bacterial type II fatty acid synthase system, ␤-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is ϳ41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K m values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 M, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC 50 value of 0.016 M. SB418011 also inhibited E. coli and H. influenzae FabH with IC 50 values of 1.2 and 0.59 M, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.Fatty acid biosynthesis in bacteria, plants and animals is carried out by the ubiquitous fatty acid synthase (FAS) 1 system. In the type I system of animals, including humans, FAS is a homodimer of two large polypeptides, each comprised of several distinct enzyme domains and an integral acyl carrier protein (ACP) (1, 2). In the type II systems of bacteria (3), plants (4), and protozoa (5), the FAS components, including the ACP, exist as discrete proteins. The corresponding enzymes of the type I and II FASs are related in structure and function but generally lack overall sequence homology. The essentiality of type II FAS for bacterial viability together with major differences between it and type I FAS suggest that broad-spectrum anti-bacterial drugs may be obtained by screening for inhibitors of the bacterial components (6 -8).In the type II FAS system, ␤-ketoacyl-ACP synthase (KAS) enzymes are central to the initiation and elongation steps and play a pivotal role in the regulation of the entire pathway (9). KAS I (FabB), II (FabF), and III (FabH) catalyze the condensation of malonyl-ACP with either acetyl-CoA (in the case of FabH) or the growing ACP-linked acyl chain to form the corresponding ␤-ketoacyl-ACP substrate for the subsequent reduction step in the elongation cycle catalyzed by FabG. FabH acts via a ping-pong mechanism (10, 11) and is unique among KAS in that it utilizes acetyl-CoA as an acyl group donor whereas FabB (12) and FabF (13) both utilize acyl-ACPs as primers. Also, unlike FabB and FabF, which are sensitive to both cerulenin and thiolactomycin (TLM), FabH is insensitive to cerulenin and much less sensitive to TLM (3,12,14,15)...

The first cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor is reported. The inhibitor was obtained by rational modification of a high throughput screening lead with the aid of a S. pneumoniae FabH homology model. This homology model was used to design analogues that would have both high affinity for the enzyme and appropriate aqueous solubility to facilitate cocrystallization studies.

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As a result of increasing drug resistance in pathogenic bacteria, there is a critical need for novel broad-spectrum antibacterial agents. As fatty acid synthesis (FAS) in bacteria is an essential process for cell survival, the enzymes involved in the FAS pathway have emerged as promising targets for antimicrobial agents. Several lines of evidence have indicated that bacterial condensing enzymes are central to the initiation and elongation steps in bacterial fatty acid synthesis and play a pivotal role in the regulation of the entire fatty acid synthesis pathway. beta-ketoacyl-acyl carrier protein (ACP) synthases (KAS) from various bacterial species have been cloned, expressed and purified in large quantities for detailed enzymological, structural and screening studies. Availability of purified KAS from a variety of bacteria, along with a combination of techniques, including combinatorial chemistry, high-throughput screening, and rational drug design based on crystal structures, will undoubtedly aid in the discovery and development of much needed potent and broad-spectrum antibacterial agents. In this review we summarize the biochemical, biophysical and inhibition properties of beta-ketoacyl-ACP synthases from a variety of bacterial species.