β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis

Bacteria synthesize fatty acids by using the type II, or dissociated, fatty acid synthase system. This biosynthetic pathway has been studied primarily in Escherichia coli, providing a fairly complete picture of the mechanisms that govern the synthesis of straight-chain saturated and unsaturated fatty acids (for reviews, see references 7, 24, and 25). The pathway consists of a collection of individual proteins encoded by unique genes that function in concert to produce the variety of fatty acid products found in bacteria. The genomes of a number of bacteria are now known, and all these organisms possess a highly related, and clearly identified, set of genes that carry out the reactions in the pathway. E. coli and many other gram-negative bacteria synthesize even-chain saturated and unsaturated fatty acids. Fatty acid synthesis is initiated by the condensation of acetyl-coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (malonyl-ACP) by β-ketoacyl-ACP synthase III, the product of the fabH gene (14, 15, 31). However, other bacteria produce such a great variety of fatty acid structures that E. coli cannot be considered typical (25). Many gram-positive organisms, such as the bacilli, staphylococci, and streptomycetes, produce odd- and even-carbon-number branched-chain fatty acids (20). These branched-chain fatty acid structures have either iso- or anteiso-methyl branches, and it is tenable to hypothesize that the FabH component in organisms like B. subtilis would be able to accept isovaleryl-CoA, isobutyryl-CoA, and 2-methylbutyryl-CoA as primers to produce these fatty acids. Thus, earlier work by Butterwork and Bloch (5), showing that acyl-CoA:ACP transacylase activity, one of the reactions catalyzed by FabH (31), in Bacillus cell extracts was selective for branched-chain acyl-CoAs is consistent with the idea that bFabH prefers these primers. Genetic experiments with B. subtilis demonstrate that these CoA thioesters are essential intermediates in fatty acid synthesis and arise from iso- and anteiso-branched α-keto acids derived from the biosynthetic pathways for the amino acids valine, leucine, and isoleucine (33, 34). The enzyme responsible for the formation of these acyl-CoAs is a specialized branched-chain α-keto acid dehydrogenase complex, and mutations in the activity of this enzyme system result in strains that are auxotrophic for branched-chain acids (34). The Streptomyces glaucescens FabH enzyme efficiently utilizes both butyryl-CoA and isobutyryl-CoA and is thought to be responsible for initiating branched-chain fatty acid synthesis in this organism (10). eFabH is selective for acetyl-CoA (12, 14, 15, 31), and its low activity with butyryl-CoA (12) suggests that bulkier branched-chain CoA thioesters may not be substrates, although this idea has not been experimentally tested. In addition to the FabH component, it is also possible that all of the enzymes of the elongation cycle in gram-negative organisms (FabF, FabG, FabZ, and FabI) cannot utilize branched-chain intermediates. The goal of this study was to biochemically characterize the FabH enzymes of B. subtilis and determine if the FabH component is unique among the enzymes of type II fatty acid synthases in its selectivity toward branched-chain intermediates.