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Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (˜ 25 °C increase in T 50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2019 Federation of European Biochemical Societies.)