Characterization of beta-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role in initiation of fatty acid biosynthesis.
The Streptomyces glaucescens fabH gene, encoding beta-ketoacyl-acyl carrier protein (beta-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an Mr of 37,000, while gel filtration analysis determined a native Mr of 72,000 +/- 3,000 (mean +/- standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a Km value of 3.66 microM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with Km values of 2.4 microM for acetyl-CoA, 0.71 microM for butyryl-CoA, and 0.41 microM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.
Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219, USA.
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D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism*; D013379:Substrate Specificity; D013876:Thiophenes / Q000494:pharmacology; D014633:Valine / Q000378:metabolism
D017931:DNA Primers; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D013876:Thiophenes; C035068:thiolactomycin; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D007930:Leucine; D014633:Valine