Structural and Functional Analysis of A-Type Ketoreductases from the Amphotericin Modular Polyketide Synthase

SummaryComplex polyketides are characterized by multiple chiral centers harboring hydroxyl and alkyl substituents. To investigate the mechanisms by which these stereocenters are set, several high-resolution structures of the ketoreductase (KR) domain from the second module of the amphotericin modular polyketide synthase (PKS) were solved. This first structural analysis of an A-type KR helps reveal how these KRs direct polyketide intermediates into their active sites from the side opposite that used by B-type KRs, resulting in a β-hydroxyl group of opposite stereochemistry. A comparison of structures obtained in the absence and presence of ligands reveals an induced fit mechanism that is important for catalysis. Activity assays of mutants of KRs from the first and second modules of the amphotericin PKS reveal the relative contributions of several active site residues toward catalysis and stereocontrol. Together, these results highlight the possibility of region-specific modification of polyketides through active site engineering of KRs.

► This first A-type KR structure helps explain how KR types set stereocenters
► The complete nicotinamide cofactor is bound and its binding site described
► The binding of ligands induces conformational changes necessary for catalysis
► A stereocontrol assay reveals mutation that reverses α-substituent stereochemistry