P. Ross Wilderman

Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner Adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1-Adx complex. We report that differential cross-linking internal to CYP24A1 point toward an Adx-induced conformational change that perturbs the F and G helices, which are required for substrate binding. Moreover, the modeled complex suggests that a semi-conserved nonpolar interaction at the interface may influence CYP24A1 regioselectivity. Taken together, these findings contribute to our understanding of Adx recognition in a critical vitamin-D inactivating enzyme as well as provide broader insight regarding the variability inherent in CYP-Adx interactions.

Although herbivory is widespread among mammals, few species have adopted a strategy of dietary specialization. Feeding on a single plant species often exposes herbivores to high doses of plant secondary metabolites (PSMs), which may exceed the animal's detoxification capacities. Theory predicts that specialists will have unique detoxification mechanisms to process high levels of dietary toxins. To evaluate this hypothesis, we compared liver microsomal metabolism of a juniper specialist, Neotoma stephensi (diet >85% juniper), to a generalist, N. albigula (diet ≤30% juniper). Specifically, we quantified the concentration of a key detoxification enzyme, cytochrome P450 2B (CYP2B) in liver microsomes, and the metabolism of α‐pinene, the most abundant terpene in the juniper species consumed by the specialist woodrat. In both species, a 30% juniper diet increased the total CYP2B concentration (2–3×) in microsomes and microsomal α‐pinene metabolism rates (4‐fold). In N. stephensi, higher levels of dietary juniper (60% and 100%) further induced CYP2B and increased metabolism rates of α‐pinene. Although no species‐specific differences in metabolism rates were observed at 30% dietary juniper, total microsomal CYP2B concentration was 1.7× higher in N. stephensi than in N. albigula (p < .01), suggesting N. stephensi produces one or more variant of CYP2B that is less efficient at processing α‐pinene. In N. stephensi, the rates of α‐pinene metabolism increased with dietary juniper and were positively correlated with CYP2B concentration. The ability of N. stephensi to elevate CYP2B concentration and rate of α‐pinene metabolism with increasing levels of juniper in the diet may facilitate juniper specialization in this species.

Although herbivores rely on liver enzymes to biotransform plant secondary metabolites ingested in plant-based diets, only a few enzymes from a handful of species have been characterized at the genomic level. In this study, we examined cytochrome P450 2B (CYP2B) sequence diversity and gene copy number in a conifer specialist, the red tree vole (Arborimus longicaudus). We fed captive individuals exclusively Douglas-fir (Pseudotsuga menziesii) foliage, cloned and sequenced their liver CYP2B cDNA, and estimated CYP2B gene copy number. We identified 21 unique CYP2B nucleotide sequences, and 20 unique CYP2B amino acid sequences. Gene copy number of CYP2B was estimated at 7.7 copies per haploid genome. We compared red tree vole CYP2B with CYP2B sequences of a generalist, the prairie vole (Microtus ochrogaster), found in GenBank. Our study revealed that the CYP2B enzymes of red tree voles possess unique sequences compared to CYP2B enzymes of other herbivorous species. The unique combination of amino acid residues at key substrate recognition sites of CYP2B enzymes may underlie the ability of the red tree vole to specialize on a highly toxic diet of Douglas-fir.

Theory postulates that dietary specialization in mammalian herbivores is enabled by a specialized set of liver enzymes that process the high concentrations of similar plant secondary metabolites (PSMs) in the diets of specialists. To investigate whether qualitative and quantitative differences in detoxification mechanisms distinguish dietary specialists from generalists, we compared the sequence diversity and gene copy number of detoxification enzymes in two woodrat species: a generalist, the white‐throated woodrat (Neotoma albigula) and a juniper specialist, Stephens’ woodrat (N. stephensi). We focused on enzymes in the cytochrome P450 subfamily 2B (CYP2B), because previous research suggests this subfamily plays a key role in the processing of PSMs. For both woodrat species, we obtained and sequenced CYP2B cDNA, generated CYP2B phylogenies, estimated CYP2B gene copy number and created a homology model of the active site. We found that the specialist possessed on average ~5 more CYP2B gene copies than the generalist, but the specialist's CYP2B sequences were less diverse. Phylogenetic analysis of putative CYP2B homologs resolved woodrat species as reciprocally monophyletic and suggested evolutionary convergence of distinct homologs on similar key amino acid residues in both species. Homology modelling of the CYP2B enzyme suggests that interspecific differences in substrate preference and function likely result from amino acid differences in the enzyme active site. The characteristics of CYP2B in the specialist, that is greater gene copy number coupled with less sequence variation, are consistent with specialization to a narrow range of dietary toxins.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a K_S) and IC₅₀ values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein–ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2′4′5′TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b₅ to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC₅₀ value and decreased the 2′4′5′TCPOA IC₅₀ value. Overall, the inconsistency between K_S and IC₅₀ values for POA versus 2′4′5′TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.

On the basis of recent functional and structural characterization of cytochromes P450 2B from the desert woodrat (Neotoma lepida), the 7-alkoxycoumarin and 7-alkoxy-4-(trifluoromethyl)coumarin O-dealkylation profiles of CYP2B35 and CYP2B37 were re-engineered. Point mutants interchanging residues at seven positions in the enzyme active sites were created and purified from an Escherichia coli expression system. In screens for O-dealkylation activity, wild-type CYP2B35 metabolized long-chain 7-alkoxycoumarins but not 7-alkoxy-4-(trifluoromethyl)coumarins or short-chain 7-alkoxycoumarins. Wild-type CYP2B37 metabolized short-chain substrates from both series of compounds. CYP2B35 A367V showed maximal activity with 7-butoxycoumarin as opposed to 7-heptoxycoumarin in the parental enzyme, and CYP2B35 A363I/A367V produced an activity profile like that generated by CYP2B37. CYP2B35 A363I/A367V/I477F showed 7-ethoxycoumarin and 7-ethoxy-4-(trifluoromethyl)coumarin O-dealkylation rates similar to those of CYP2B37 and higher than those of the double mutant. A CYP2B35 septuple mutant retained a CYP2B37-like activity profile. In contrast, the CYP2B37 septuple mutant produced very low rates of O-dealkylation of all substrates. As mutating residue 108 in either enzyme was detrimental, this change was removed from both septuple mutants. Remarkably, the CYP2B35 sextuple mutant produced an activity profile that was a hybrid of that of CYP2B35 and CYP2B37, whereas the CYP2B37 sextuple mutant had almost no O-dealkylation activity. Docking of 7-substituted coumarin derivatives into a model of the CYP2B35 sextuple mutant based on a previous crystal structure of the 4-(4-chlorophenyl)imidazole wild-type complex revealed how the mutant can exhibit activities of both CYP2B35 and CYP2B37.

Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structure-function relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates—namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarins—with a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (k_cat/K_M) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B–4-CPI–bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple –Cl and –NH-π interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.

Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH₃, or -CF₃ at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.