Structural studies of an A2-type modular polyketide synthase ketoreductase reveal features controlling α-substituent stereochemistry

摘要

Modular polyketide synthase ketoreductases often set two stereocenters when reducing intermediates in the biosynthesis of a complex polyketide. Here we report the 2.60 Å-resolution structure of an A2-type ketoreductase from the eleventh module of the amphotericin polyketide synthase that sets a combination of l-α-methyl and l-β-hydroxyl stereochemistries and represents the final catalytically-competent ketoreductase type to be structurally elucidated. Through structure-guided mutagenesis a double mutant of an A1-type ketoreductase was generated that functions as an A2-type ketoreductase on a diketide substrate analog, setting an α-alkyl substituent in an l- orientation rather than in the d-orientation set by the unmutated ketoreductase. When the activity of the double-mutant was examined in the context of an engineered triketide lactone synthase, the anticipated triketide lactone was not produced even though the ketoreductase-containing module still reduced the diketide substrate analog as expected. These findings suggest that re-engineered ketoreductases may be catalytically outcompeted within engineered polyketide synthase assembly lines.