44399-G1
Regiospecific Synthesis of Polyketide Natural Products Using Polyketide Synthase
Sheryl Tsai, University of California, Irvine
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The Type II polyketide synthase (PKS) is an enzyme complex comprised of 5 – 10 distinct domains that produce pharmaceutically important natural products. Polyketide diversity is achieved via a controlled variation of extender units, chain length, cyclization and reduction patterns. During the past decade, a body of knowledge has been well-developed on the cloning, genetic analysis and product characterization of PKS. However, there is a knowledge gap in correlating the PKS structures with protein-ligand interactions, enzyme catalysis, and substrate specificity. Such a knowledge gap has severely hampered protein engineering efforts of PKS. Without information about sequence-structure-function relationships within and among PKS domains, random shuffling of PKS domains often results in the complete loss of enzyme activity. Further, although more than 50 PKS enzyme clusters have been cloned, there is little information that correlates protein sequence with different natural product outcome, due to the lack of detailed structure-function studies. Therefore, there is a need for the structural determination of individual PKS domains, as well as a correlation of their sequences and functions to the observed biological activities. We have made significant progress on elucidating the sequence-structure-function relationship of two aromatic PKS domains, the ketoreductase (KR) and bifunctional aromatase/cyclase (ARO/CYC). KR and ARO/CYC catalyze the polyketide chain reduction and cyclization, respectively, in a highly specific manner. Based on 14 crystal structures of KR and ARO/CYC, > 50 structure-based mutants, five in vitro substrates and kinetic studies of the Type II PKS, specifically, on the actinorhodin KR (actKR) and tetracenomycin ARO/CYC (tcmARO), we have conducted structure-directed mutagenesis and successfully changed the substrate specificity and enzyme activity in a predictable manner. This has allowed us to reach the proposed research goal for the Petroleum Research Fund in biosynthesizing new petroleum related polyketide products with rationalized control of chain length, reduction and cyclization patterns for the first time.
