Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Session: 2
Caylie McGlade - Winthrop University
Co-Author(s): Rosa Vasquez, University of Michigan, Ann Arbor, MI; Kinshuk Srivastava, University of Michigan, Ann Arbor, MI; Sean Newmister, University of Michigan, Ann Arbor, MI; Jessica Stachowski, University of Michigan, Ann Arbor, MI; John Montgomery, University of Michigan, Ann Arbor, MI; David Sherman, University of Michigan, Ann Arbor, MI
Many biologically active molecules are oxidized hydrocarbons. For this reason, there has been much research conducted on functionalizing unactivated hydrocarbons. Traditional methods of C-H functionalization (such as transition-metal catalysts), while effective, require harsh conditions and additional protecting and deprotecting steps. Conversely, enzymes are able to functionalize C-H bonds under mild conditions and with exquisite regio- and stereoselectivity. One class of enzymes, called cytochromes P450, are able to catalyze oxidations on unfunctionalized hydrocarbons. These enzymes are typically monooxygenases, capable of catalyzing only a single oxidation, although there have been some plant and fungal P450s that have been found to catalyze more than three consecutive oxidations. However, these membrane P450s are difficult to study due to poor solubility.
A new P450, TamI, has been recently isolated from a marine Streptomyces species. This enzyme is able to catalyze up to three oxidations in the biosynthetic pathway of the antibiotic tirandamycin. The selective oxidative mechanism of TamI is not yet understood. In this study, we hypothesized that we would be able to gain a deeper understanding of the selectivity displayed by TamI via site-directed mutagenesis, as well as expand the substrate scope of the enzyme to recognize different cyclic and bicyclic moieties, potentially leading to the development of more effective antibiotics. Site-directed mutagenesis was accomplished by targeting specific residues for mutation (guided by a high-resolution x-ray structure of TamI complexed with its natural substrate tirandamycin C), designing primers, and introducing mutations by PCR. Once the protein was overexpressed and purified, enzymatic reactions were conducted and analyzed using RP-HPLC and LC-MS. Only two of the four newly targeted mutants, G248A and T252A, were obtained and gave no new selectivity. One interesting previously targeted mutant, L101A, yielded three new tirandamycin derivatives. This TamI mutant protein was harvested for preparative-scale enzymatic reactions with tirandamycin C to isolate and characterize the reaction products. Purification of these products by RP-HPLC is in progress. In addition, synthesis of two tirandamycin-like substrates was accomplished with a Yamaguchi coupling of unnatural anchor groups. The products of these reactions were obtained in good yield (~30-50%) without optimization. Preliminary enzymatic reactions with one unnatural substrate and a TamI mutant yielded three single oxidation products, indicating that expanding the substrate scope of TamI may be feasible. Future work will be focused on continuing to synthesize new unnatural substrates and obtaining and testing new TamI mutants.
Funder Acknowledgement(s): Funds for this research were provided by the National Science Foundation Award No. CHE- 1700982.
Faculty Advisor: Dr. David Sherman, davidhs@umich.edu
Role: I designed the primers for the new TamI mutants, overexpressed the successful mutations, and purified them. I also performed the enzymatic reactions of the new mutants and analyzed them with RP-HPLC and LC-MS. I assisted with the preparative-scale enzymatic reactions of the L101A TamI mutant. I also assisted with the synthesis of the two new tirandamycin-like substrates and the preliminary enzymatic reactions performed with one of them.