Rational evolution of the cofactor‐binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes

Abstract

NADPH‐cytochrome P450 reductase (CPR) is the natural redox partner of microsomal cytochrome P450 enzymes. CPR shows a stringent preference for NADPH over the less expensive cofactor, NADH, economically limiting its use as a biocatalyst. The complexity of cofactor‐linked CPR protein dynamics and the incomplete understanding of the interaction of CPR with both cofactors and electron acceptors present challenges for the successful rational engineering of a CPR with enhanced activity with NADH. Here, we report a rational evolution approach to enhance the activity of CPR with NADH, in which mutations were introduced into the NADPH‐binding flavin adenine dinucleotide (FAD) domain. Multiple CPR mutants that used NADH more effectively than the wild‐type CPR in the reduction of the surrogate electron acceptor, cytochrome c were found. However, most were inactive in supporting P450 activity, arguing against the use of cytochrome c as a surrogate electron acceptor. Unexpectedly, several mutants showed significantly improved activity towards CYP2C9 (mutant 1‐014) and/or CYP2A6 (mutants 1‐014, 1‐015, 1‐053 and 1‐077) using NADPH, even though the mutations were introduced at locations remote from the putative CPR‐P450 interaction face. Therefore, mutations at sites in the FAD domain of CPR may be promising future engineering targets to enhance P450‐mediated substrate turnover. Enzymes NADPH‐cytochrome P450 reductase – EC http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/6/2/4.html; cytochrome P450 – EC http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/14/14/1.html.

Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts

“…Under these conditions, this variant was found to produce the desired sultam product 1b in 70% yield with a TTN value of 14 800 (Table , Entry 17). This TTN value corresponds to the highest catalytic activity for a biocatalytic nitrene transfer reaction reported to date, with only one notable exception, and it compares well with the highest TTN values reported for monooxygenation reactions catalyzed by engineered P450s. − …”

“…Arnold and co-workers reported a comparable activity (∼360 TON) for serine-ligated P450 BM3 variants in the same reaction, and a similar performance was observed for artificial P450 metalloenzymes harboring an abiological Ir­(Me) mesoporphyrin IX cofactor (∼300 TON) . While the catalytic activity of these engineered P450s in C–H amination reactions exceeds that of synthetic catalysts (typically, <100–150 TON), this reactivity remains far below that achievable by these enzymes in monooxygenation reactions, some of which have been reported to exceed 60 000 total turnovers. − …”

Across kingdom biased CYP-mediated metabolism via small-molecule ligands docking on P450 oxidoreductase

“…Docking of structurally diverse ligands biases GPCR conformational sampling, stabilizing distinct conformational states and thus the corresponding signaling pathways, a phenomenon called biased agonism [15][16][17] . POR acts as a metabolic hub: Its conformational sampling and specificity towards CYPs is dependent on regulatory cues 5,6,18 and mutations 4,19 indicating that POR specificity of activating metabolic cascades may operate via mechanisms akin to biased agonism. However, to date no small molecules are known to target metabolic hubs like POR and allosterically control downstream metabolic pathways.…”

“…Furthermore, it shows that direct docking on POR should be taken into consideration when screening for drug-CYP interactions. Supplementary Fig 2 and Given that point-mutations in human POR can lead to altered specificity towards CYP isoforms 4, 19 , we tested whether small-molecule ligands can bias the specificity of human POR to reduce diverse electron acceptors. Comparative in vitro activity assays were carried out using commonly employed artificial electron acceptors of POR; Cytc 23,24 , resazurin (RS) 25 or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 26 (Fig 2A).…”

Across kingdom biased CYP-mediated metabolism via small-molecule ligands docking on P450 oxidoreductase

“…Furthermore, it shows that direct docking on POR should be taken into consideration when screening for drug-CYP interactions. Supplementary Fig 2 and Given that point-mutations in human POR can lead to altered specificity towards CYP isoforms 4, 19 , we tested whether small-molecule ligands can bias the specificity of human POR to reduce diverse electron acceptors. Comparative in vitro activity assays were carried out using commonly employed artificial electron acceptors of POR; Cytc 23,24 , resazurin (RS) 25 or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 26 (Fig 2A).…”