Identification, Substrate Specificity, and Inhibition of theStreptococcus pneumoniae β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH)

Abstract

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)...

2,5-Dialkylresorcinol Biosynthesis in Pseudomonas aurantiaca : Novel Head-to-Head Condensation of Two Fatty Acid-Derived Precursors

“…For example, both E. coli and Streptococcus pneumoniae synthesize only straight-chain fatty acids. The corresponding FabH enzymes for these bacteria accept acetylCoA, propionyl-CoA, and, to a small extent, butyryl-CoA (but not long-or branched-chain acyl-CoA) primers, despite the apparent sequence divergence between the substrate binding pockets of the two enzymes (33). In contrast, FabH from Staphylococcus aureus, which produces a relatively high percentage of branched-chain fatty acids, accepts acetyl-CoA and butyryl-CoA as primers, but the most preferred substrate is isobutyryl-CoA.…”

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

“…Acetyl-CoA carboxylase plays a critical role in controlling fatty acid metabolism. Expression of the fabH gene, encoding a ␤-ketoacyl-acyl carrier protein synthase III gene which catalyzes the condensation of malonylacyl carrier protein and an acyl-CoA substrate (32), was increased in the less-tolerant 824(pSOS95del) strain and lower in the more-tolerant 824(pGROE1) strain. FabH has been shown to be an important determinant of branched-chain fatty acid synthesis (8) in B. subtilis.…”

Purification and Biochemical Characterization of theMycobacterium tuberculosis β-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

“…Distinct roles for KasA and KasB in mycolic acid synthesis were postulated based upon increases in the incorporation of radioactivity into fatty acids in various FAS assays after overexpression of either kasA or kasB (25). The antimycobacterial activity of TLM suggests the essentiality of at least one of the three condensing enzymes, as is the case for orthologs in other bacterial species such as E. coli FabB (26) and Streptococcus pneumoniae FabH (27) and FabF, 3 thus making them attractive targets for the development of screens to identify new lead compounds. Therefore, we purified and characterized KasA and KasB, and despite many similarities, these enzymes display differing enzymatic properties and may indeed play distinct roles in mycolic acid biosynthesis.…”