Monday, August 2, 2010
Pacific Concourse (Hyatt Regency San Francisco)
Pouya Javidpour, Tyler P. Korman and Shiou-Chuan Tsai, Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA
Type II polyketides are a large class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic oxytetracycline and antitumor agent doxorubicin. The type II polyketide synthase (PKS) consists of 5-10 dissociated domains and is homologous to fatty acid synthase (FAS). The ketoreductase (KR) enzyme regiospecifically reduces the C9-carbonyl group and has the potential to introduce stereochemistry in the polyketide substrate. The molecular basis for how the type II KR achieves such specificity is not well understood. A comparison of actinorhodin (act) and hedamycin (hed) ketoreductases with related SDR proteins shows that a well-conserved 94-XGG-96 (actKR numbering) motif may determine stereospecificity of reduction. Single, double, and triple actKR mutant combinations of P94L, G95D, and G96D were generated and analyzed through in vitro kinetic assays and characterization of polyketides produced in vivo. The results show that the actKR P94L single mutation is sufficient to switch the in vitro preference for the S over the R stereomer from 3:1 in wild type to an exclusive preference for the S stereomer. Crystal structures of the P94L-NADPH and P94L-NADPH-emodin complexes, together with structural and kinetic analyses of hedamycin KR, suggest that stereospecificity in type II KR is determined by steric effects imposed by pocket shape. The results pave the foundation toward the application of type II KR for stereospecific chemoenzymatic synthesis.
