Exploration of the Diverse Functions of Cytochrome P450 Monooxygenases Towards the Development of Biocatalysts.

Abstract

The superfamily of cytochrome P450 monooxygenases is involved in diverse oxidative processes including xenobiotic detoxification, steroid synthesis, and biosynthetic tailoring of diverse secondary metabolites. Members of this superfamily catalyze a vast range of reactions from hydroxylations to isomerizations and ring formations. Given the ease of cloning, protein overexpression, and purification, bacterial biosynthetic P450s have garnered special interest given their catalytic efficiency and high regio- and stereoselectivity. Nevertheless, barriers still exist to the practical application of P450s, including instability, dependence on redox partners, and limited substrate scope. My dissertation has focused on investigating P450s as potential biocatalysts for the production of pharmaceuticals. First, a substrate-engineering approach elaborated on a preexisting hypothesis revealing unprecedented flexibility of an engineered chimeric P450, PikCD50N-RhFRED, previously developed. We gained valuable insight into the fundamental factors affecting P450-mediated oxidation, such as substrate binding, orientation, and product formation. The results culminated in an optimized linear linker series, effectively replacing the natively used anchor. These advancements eliminated labor-intensive synthetic steps, opening to the door to facile chemoenzymatic elaboration. To further expand upon PikCD50N-RhFRED research, the chimera was employed to selectively oxidize structurally distinct scaffolds with pharmaceutical applications, including tamoxifen and tiamulin. These results underscored PikC’s flexibility and potential as a biocatalyst through exploiting a terminal N,N-dimethylamino. Furthermore, the regioselective oxidation of these compounds highlighted the potential use of this enzyme towards predictive production of in vivo metabolites and development of drug analogs. Finally, two P450s from the tylosin biosynthetic pathway, TylI and TylHI, were functionally characterized. The enzymes were utilized as chimeras with RhFRED to afford single component, self-sufficient P450s. Using substrates isolated from fermenting Streptomyces fradiae mutants, TylI-RhFRED and TylHI-RhFRED were confirmed to be P450 hydroxylases and their sites of oxidation determined through structural elucidation. TylI-RhFRED was especially interesting given its ability to perform sequential oxidations to form an aldehyde crucial to tylosin’s bioactivity. However, this study also leaves a number of unanswered questions for future exploration of these unique P450s, mainly the method of substrate recognition employed by these two biosynthetic P450s and the source of the inherent flexibility of a P450 to perform sequential oxidations.