Structural modeling and site-directed mutagenesis of the actinorhodin beta-ketoacyl-acyl carrier protein synthase

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