Engineering a Hyper‐Fluorescent NADPH:Cytochrome P450 Oxidoreductase

Abstract

In order to study molecular interactions in the microsomal cytochrome P450 monooxygenase pathway, we have undertaken the development of a NADPH:cytochrome P450 oxidoreductase (CYPOR) with highly enhanced flavin fluorescence while maintaining wild-type (WT) structure and function. Using a bacterial expression system, a soluble human CYPOR lacking the N-terminal 66 residues was produced, comprised of the catalytic FMN-, FAD- and NADPH-binding domains. Using site-directed mutagenesis of our expression plasmid, we have created six variants of human CYPOR; three having substitutions in the FMN-binding domain (Y143F, Y143A, and Y143S) and three in the FAD-binding domain (Y459F, Y459A, and Y459S). All six variants, as well as WT, were expressed and purified to homogeneity. Flavin analysis showed that Y143 variants had slightly decreased FMN content, while Y459 variants had dramatically reduced FAD content, compared to WT. Y143A and Y143S variants exhibited 3.5- and 6.5-fold increases in flavin fluorescence compared to WT, respectively. The Y143S variant retained 85% of WT NADPH:cytochrome c reductase activity, while Y143A was 70% more active than WT. Based on the observed increase in fluorescence and on the retained catalytic efficiency, Y143 variants currently represent the best candidates for development as tools for studying CYPOR interactions with P450s and other microsomal proteins. With proof of principal established, future constructs will incorporate the membrane anchoring residues, for reconstitution of cytochrome P450-mediated activities in vitro and in cellular expression systems (Supported by NIH HL30050 to BSM).