Biochemical, structural, and bioengineering studies of cytochrome P450 enzymes involved in biosynthesis of secondary metabolites

The superfamily of cytochrome P450 monooxygenases is involved in diverse oxidative processes including xenobiotic catabolism, steroid synthesis, and biosynthetic tailoring of diverse natural products. During the past decade, the synthetic potential of biosynthetic P450 enzymes from microorganisms has gained special attention due to their non-membrane bound nature, considerable catalytic efficiency, and high regio- and stereoselectivity. However, current barriers to their application in synthetic chemistry include their instability, inherent dependence on separate redox partners, and narrow substrate spectra. As these hurdles have been gradually overcome, it is likely that these biosynthetic P450s will find expanded use in the production of chemicals, fragrances, pharmaceutical compounds, biofuels, and application in bioremediation.;My dissertation research has focused on the bacterial cytochrome P450 PikC from the pikromycin macrolide antibiotic biosynthetic pathway in Streptomyces venezuelae. The inherent substrate flexibility and hydroxylation pattern of PikC suggests its unique oxidative mechanism and synthetic potential. Starting from the crystal structures of PikC, we not only elucidated the structural basis for its substrate flexibility, but also discovered a unique desosamine sugar anchoring functionality of this enzyme. These observations directly inspired a substrate engineering strategy that utilizes the desosamine anchor to deliver diverse structures into the PikC active site for selective oxidation. Using this approach, the substrate spectrum of PikC has been significantly broadened. Specifically, by using an engineered PikCD50N -RhFRED with self-sufficiency and significantly higher catalytic efficiency, a series of carbocyclic rings linked to the desosamine glycoside were effectively hydroxylated in a regioselective manner Associated analysis of co-crystal structures of PikC with selected unnatural desosaminyl substrates provided significant insights into the mechanism of its oxidative selectivity control. Taken together, these results offer an applicable enzymatic solution of a central challenge in synthetic chemistry—the selective oxidation of an unactivated sp3 C-H bond.;Moreover, a number of other biosynthetic P450 enzymes, two O -methyltransferases, an FAD-dependent oxidase, and a type III polyketide synthase were also studied during the course of my dissertation research. Together, these studies provide new insights into biosynthesis of secondary metabolites and how these enzymes can be adapted for biotechnological use.