Characterization of beta-ketoacyl-acyl carrier protein synthase III from Streptomyces glaucescens and its role in initiation of fatty acid biosynthesis.
نویسندگان
چکیده
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.
منابع مشابه
Characterization of b-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis
متن کامل
Beta-ketoacyl acyl carrier protein synthase III (FabH) is essential for fatty acid biosynthesis in Streptomyces coelicolor A3(2).
The Streptomyces coelicolor fab (fatty acid biosynthesis) gene cluster (fabD-fabH-acpP-fabF) is cotranscribed to produce a leaderless mRNA transcript. One of these genes, fabH, encodes a ketoacyl synthase III that is essential to and is proposed to be responsible for initiation of fatty acid biosynthesis in S. coelicolor.
متن کاملEngineered fatty acid biosynthesis in Streptomyces by altered catalytic function of beta-ketoacyl-acyl carrier protein synthase III.
The Streptomyces glaucescens beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Thre...
متن کاملb-Ketoacyl Acyl Carrier Protein Synthase III (FabH) Is Essential for Fatty Acid Biosynthesis in Streptomyces coelicolor A3(2)
Streptomyces spp. synthesize the majority of their fatty acids from branched starters such as isobutyryl, isovaleryl, and anteisovaleryl units to give oddand even-numbered fatty acids with a methyl branch at the v-terminus (80 to 90% of total fatty acid content); the remainder are synthesized from straight starters such as acetyl and butyryl units (11, 21). The fatty acid synthase (FAS) of Stre...
متن کاملMalonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomyces glaucescens: a possible link between fatty acid and polyketide biosynthesis.
Streptomyces glaucescens, a Gram-positive soil bacterium, produces the polyketide antibiotic tetracenomycin (Tcm) C. To study possible biochemical connections between the biosynthesis of bacterial fatty acids and polyketides, the abundant acyl carrier protein (ACP) detected throughout the growth of the tetracenomycin (Tcm) C-producing S. glaucescens was purified to homogeneity and found to beha...
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عنوان ژورنال:
- Journal of bacteriology
دوره 180 17 شماره
صفحات -
تاریخ انتشار 1998