Laboratory Evolution of Cytochrome P450 Peroxygenase Activity

Citation

Cirino, Patrick Carmen (2004) Laboratory Evolution of Cytochrome P450 Peroxygenase Activity. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/KWGR-DF47. https://resolver.caltech.edu/CaltechETD:etd-06062003-164310

Abstract

The ability of the cytochrome P450 heme monooxygenases to catalyze difficult oxidation reactions, often with high specificity and selectivity, makes them attractive for numerous biotechnological applications. However they are generally limited by low turnover rates and low stability, and their minimum requirements for catalysis include a cofactor as source of electrons (NAD(P)H), partner proteins for electron transfer, and dioxygen. Some P450s are capable of supporting low levels of peroxygenase activity, in which a peroxide is utilized to drive catalysis via a "shunt" pathway. This mechanism for substrate oxidation, although inefficient and not generally utilized in nature, simplifies P450 catalysis by eliminating the need for NAD(P)H.

Our goal was to engineer an efficient P450 peroxygenase which utilizes hydrogen peroxide (H₂O₂). Directed evolution is a powerful enzyme engineering methodology which mimics nature's algorithm for evolution. Enzyme libraries are generated via DNA mutagenesis or recombination techniques, and variants with improved function are isolated using an appropriate screen or selection. Using this strategy, in combination with site-directed mutagenesis, we have created P450 BM-3 heme domain variants with more than 100-fold improved H₂O₂-driven hydroxylation activity compared to wild-type, showing both an improved k_cat as well as a lower K_m for H₂O₂. Thermostability was also improved by directed evolution.

We have engineered a cell-free, biomimetic hydroxylase that requires only H₂O₂ to exploit the hydroxylating power of P450 BM-3. Peroxide-mediated inactivation as a result of heme destruction remains a major obstacle and presents an important enzyme engineering challenge. This research has broadened the potential applications of P450 biocatalysis by exploiting the versatility of heme-containing proteins.

Thesis Files