Biochemical characterization of branched chain fatty acid biosynthesis
Date
1997
Authors
Lahiri, Devlina
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Nikolau, Basil J.
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Abstract
Consistent with the fact that Escherichia coli synthesizes straight chain fatty acids, extracts from this organism can utilize acetyl-CoA, but not isovaleryl-CoA, to prime a single cycle of fatty acid biosynthesis. In contrast, extract of Bacillus subtilis, an organism that synthesizes methyl-branched fatty acids, can utilize both acetyl-CoA and isovaleryl-CoA as a primer for a single cycle of fatty acid biosynthesis. These observations indicate that B. subtilis contains a novel 13 ketoacyl-acyl carrier protein synthase that can initiate branched chain fatty biosynthesis using isovalerylCoA as a substrate. This enzyme, which we call 13-ketoacyl-acyl carrier protein synthase IIIB was purified to homogeneity by a three step procedure. The molecular weight of the protein is 44,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme is highly specific for isovaleryl CoA; Kₘ for isovaleryl-CoA was 35.8 µM. Acetyl CoA could not substitute for the branched chain substrates. This specificity of KAS IIIB suggests it has a role in catalyzing the initial reaction of branched chain fatty acid biosynthesis.
The biosynthesis of fatty acid requires the action of three condensing enzymes, β-ketoacyl-ACP synthase I, II and Ill. β-ketoacyl-ACP synthase Ill catalyzes the first condensation reaction of this process in Escherichia coli and this is encoded by the fabH gene. Using a recombination-based procedure we have disrupted the fabH gene of E.coli. The resulting strain can grow, although at a reduced rate. The exogenous supply of butyrate but not valerate or isovalerate alleviates this reduced growth rate. These results indicate that the fabH gene is not essential and that the deficiency in β-ketoacyl-ACP synthase Ill can be partially overcome by the action of β-ketoacyl-ACP synthase I.
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