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Cytochrome P450 (P450) enzymes dominate steroid metabolism. In general, the simple C-hydroxylation reactions are mechanistically straightforward and are generally agreed to involve a perferryl oxygen species (formally FeO3+). Several of the steroid transformations are more complex and involve C-C bond scission. We initiated mechanistic studies with several of these (i.e., 11A1, 17A1, 19A1, and 51A1) and have now established that the dominant modes of catalysis for P450s 19A1 and 51A1 involve a ferric peroxide anion (i.e., Fe3+O2¯) instead of a perferryl ion complex (FeO3+), as demonstrated with 18O incorporation studies. P450 17A1 is less clear. The indicated P450 reactions all involve sequential oxidations, and we have explored the processivity of these multi-step reactions. P450 19A1 is distributive, i.e., intermediate products dissociate and reassociate, but P450s 11A1 and 51A1 are highly processive. P450 17A1 shows intermediate processivity, as expected from the release of 17-hydroxysteroids for the biosynthesis of key molecules, and P450 19A1 is very distributive. P450 11B2 catalyzes a processive multi-step oxidation process with the complexity of a chemical closure of an intermediate to a locked lactol form. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450, CYP) 19A1 is the steroid aromatase, the enzyme responsible for the 3‐step conversion of androgens (androstenedione or testosterone) to estrogens. The final step is C−C bond scission (removing the 19‐oxo group as formic acid) that proceeds via a historically controversial reaction mechanism. The two competing mechanistic possibilities involve a ferric peroxide anion (Fe3+O2−, Compound 0) and a perferryl oxy species (FeO3+, Compound I). One approach to discern the role of each species in the reaction is with the use of oxygen‐18 labeling, i.e. from 18O2 and H218O of the reaction product formic acid. We applied this approach, using several technical improvements, to study the deformylation of 19‐oxo‐androstenedione by human P450 19A1 and of a model secosteroid, 3‐oxodecaline‐4‐ene‐10‐carboxaldehyde (ODEC), by rabbit P450 2B4. Both aldehyde substrates were sensitive to non‐enzymatic acid‐catalyzed deformylation, yielding 19‐norsteroids, and conditions were established to avoid issues with artifactual generation of formic acid. The Compound 0 reaction pathway predominated (i.e. Fe3+O2−) in both P450 19A1 oxidation of 19‐oxo‐androstenedione and P450 2B4 oxidation of ODEC. The P450 19A1 results contrast with our prior conclusions (J. Am. Chem. Soc. 2014, 136, 15016–16025), attributed to several technical modifications. [ABSTRACT FROM AUTHOR]

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.

The 14α‐demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three‐step reaction terminates in a C−C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2−) or Compound I (perferryl oxygen, FeO3+) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18O incorporation into formic acid from 18O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18O (one atom) into formic acid, consistent with a major but not exclusive FeO2− mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X‐ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Å away from the heme iron atom. Experiments with human P450 51A1 and H218O yielded primarily one 18O atom but 14 % of the formic acid product with two 18O atoms, indicative of a minor contribution of a Compound I mechanism. LC–MS evidence for a Compound 0‐derived Baeyer–Villiger reaction product (a 14α‐formyl ester) was also found. [ABSTRACT FROM AUTHOR]

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.