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Journal of Bacteriology, May 2000, p. 2619-2623, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Structural Modeling and Site-Directed Mutagenesis of the Actinorhodin beta -Ketoacyl-Acyl Carrier Protein Synthase

Min He, Mustafa Varoglu, and David H. Sherman*

Department of Microbiology and Biological Process Technology Institute, University of Minnesota, Minneapolis, Minnesota 55455

Received 10 December 1999/Accepted 9 February 2000

A three-dimensional model of the Streptomyces coelicolor actinorhodin beta -ketoacyl synthase (Act KS) was constructed based on the X-ray crystal structure of the related Escherichia coli fatty acid synthase condensing enzyme beta -ketoacyl synthase II, revealing a similar catalytic active site organization in these two enzymes. The model was assessed by site-directed mutagenesis of five conserved amino acid residues in Act KS that are in close proximity to the Cys169 active site. Three substitutions completely abrogated polyketide biosynthesis, while two replacements resulted in significant reduction in polyketide production. 3H-cerulenin labeling of the various Act KS mutant proteins demonstrated that none of the amino acid replacements affected the formation of the active site nucleophile.

* Corresponding author. Mailing address: Department of Microbiology, Box 196, 1460 Mayo Memorial Building, 420 Delaware St. S.E., Minneapolis, MN 55455-0312. Phone: (612) 626-0199. Fax: (612) 624-6641. E-mail: david-s{at}biosci.umn.edu.

Journal of Bacteriology, May 2000, p. 2619-2623, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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