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Rational evolution of the cofactor‐binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes
Author(s) -
Strohmaier Silja J.,
Huang Weiliang,
Baek JongMin,
Hunter Dominic J. B.,
Gillam Elizabeth M. J.
Publication year - 2019
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14982
Subject(s) - cofactor , cytochrome p450 reductase , cytochrome p450 , reductase , biochemistry , mutant , enzyme , chemistry , biology , cytochrome c , coenzyme q – cytochrome c reductase , gene , mitochondrion
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 1.6.2.4 ; cytochrome P450 – EC 1.14.14.1 .