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Journal of Bacteriology, September 1998, p. 4481-4486, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Characterization of beta -Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

Lei Han,dagger Sandra Lobo, and Kevin A. Reynolds*

Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219

Received 9 March 1998/Accepted 17 June 1998

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 µM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with Km values of 2.4 µM for acetyl-CoA, 0.71 µM for butyryl-CoA, and 0.41 µM 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.

* Corresponding author. Mailing address: ISBDD, Suite 212B, 800 East Leigh St., Virginia Commonwealth University, Richmond, VA 23219. Phone: (804) 828-5679. Fax: (804) 827-3664. E-mail: kareynol{at}hsc.vcu.edu.

dagger Present address: Biological Process Technology Institute and Department of Microbiology, University of Minnesota, St. Paul, MN 55108.

Journal of Bacteriology, September 1998, p. 4481-4486, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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