Engineering a Library of Light Activated P450 Biocatalysts

The cytochromes P450 (CYP) are a superfamily of heme-thiolate enzymes that utilize molecular dioxygen and two reducing equivalents to catalyze the oxidation of a C-H bond in a wide variety of organic molecules with high degree of regio and stereo selectivity.  In vivo, they are involved in the metabolism of drugs, toxins, and steroid compounds. Due to their synthetic potential, these enzymes have been of great interest for biotechnological applications.  Our research laboratory has been interested in an alternative, light-activated, approach to deliver the necessary electrons to the heme domain using hybrid P450-BM3 enzymes. The hybrid enzymes are composed of a photosensitizer covalently attached to non-native single cysteine mutants of P450 heme domains. We have previously demonstrated that these hybrid enzymes are capable of performing the hydroxylation of lauric acid with total turnover numbers of 80. We are currently developing a library of hybrid enzymes by varying the properties of the enzyme heme domain. We are using site-directed mutagenesis to systematically vary the position of the non-native single cysteine residue as well as to modify the substrate recognition properties of the heme domain. The proper mutations are confirmed by DNA sequencing.  After transformation into E. coli strain BL21(DE3), the proteins are expressed and purified by FPLC. After covalent attachment of the photosensitizers and further purification, the catalytic activity of the hybrid enzymes are tested under constant visible light irradiation. The same methodology has recently been applied to another member of the P450 superfamily, P450Cam.