title: Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase.
abstract: Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C-C bond formation. S315 played a role in coupling decarboxylation to C-C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.
authors.affiliation: Departments of Chemistry and Chemical Engineering, Stanford University , Stanford, California 94305, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Robbins
authors.affiliation: Departments of Chemistry and Chemical Engineering, Stanford University , Stanford, California 94305, United States.
authors.forename: Joshuah
authors.initials: J
authors.lastname: Kapilivsky
authors.affiliation: Department of Chemistry, Brown University , Providence, Rhode Island 02912-9108, United States.
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.affiliation: Departments of Chemistry and Chemical Engineering, Stanford University , Stanford, California 94305, United States.
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D017124:Conserved Sequence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D002264:Carboxylic Acids; D048630:Polyketide Synthases