New Reactions and Products Resulting from Alternative Interactions between the P450 Enzyme and Redox Partners

Abstract

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

“…3A). These results highlight the significance of the selection of redox partners when reconstituting the activity of a P450 enzyme (22). We also examined the catalytic activity of CYP-sb21 using 200 M H 2 O 2 as the sole electron and oxygen donor.…”

“…The gene encoding CYP-sb21 was amplified from pET28b-cypsb21-RhFRED by using primer set CYP-sb21-F/CYP-sb21-R2. The Rh-FRED-fdx gene fragment was amplified from pET28b-RhFRED-fdx (22) by using primer pair RhFRED-Fdx-F/RhFRED-Fdx-R. The two PCR products with designed overlap sequences were mixed, annealed, extended, and finally amplified with primer pair CYP-sb21-F2/RhFREDFdx-R, resulting in the fused cyp-sb21-RhFRED-fdx gene, which was subcloned into pET-28b via the NdeI and HindIII restriction sites to obtain pET-28b-cyp-sb21-RhFRED-fdx.…”

“…However, the optimal (either native or heterologous) redox partners often remain unknown or are difficult to express for many potential industrial P450s. Thus, it often becomes necessary to construct hybrid P450 reaction systems by using surrogate redox partner proteins in order to achieve desired catalytic processes (22).…”

“…NCIMB 9784, and a hybrid Rhodococcus-spinach reductase, RhFRED-Fdx (22). By using optimal cyanobacterial redox partners, a number of in vitro reaction factors was optimized to improve the overall rate of conversion of CsA to CsA-4-OH, such as the cosolvent and the NADPH regeneration system.…”

Strategies for the construction of insect P450 fusion enzymes

“…A photometric heme substrate-binding test was then carried out to determine whether the substrate bound to the catalytic heme domain of the purified fusion enzyme. There was no evidence of heme-substrate interaction, which may indicate poor access to the substrate-binding site resulting in the absence of a catalytically productive binding mode [72].…”