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.

Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~40°C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Non-standard abbreviations: BAIB, (diacetoxyiodo)benzene; CPR, cytochrome P450 reductase; CuOOH, cumene hydroperoxide; F-BAIB, bis(trifluoroacetoxy)iodobenzene; hCPR, human NADPH-cytochrome P450 reductase; LSF, late stage functionalization; luciferin H-EGE, ethylene glycol ester of 6'-desoxyluciferin; luciferin ME-EGE, ethylene glycol ester of luciferin 6'-methyl ether; luciferin MultiCYP, methyl ester of luciferin 6'methyl ether; NGS, NADPH-generating system; OS, oxygen surrogate; P450, cytochrome P450, heme-thiolate protein P450; PhIO, iodosylbenzene; tert-BuOOH , tert-butyl hydroperoxide.

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.