Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism

Abstract

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 structurefunction 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-methylcoumarinswith 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-p 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.

Structure of Cytochrome P450 2C9*2 in Complex with Losartan: Insights into the Effect of Genetic Polymorphism

“…Of note, several amino acids on the C-D loop (~135 to 140) near the Arg144 region are not consistently observed in many CYP structures due to disorder in electron density. Thus, this loop region is often not modelled with amino acids in many CYP crystal structures, due to disordered or missing electron density resulting from the crystallographic data (Shah et al, 2016;Shah et al, 2011). This was consistent in the current CYP2C9*2 complex where the residues from 135-138 were not modelled due to disorder in several chains.…”

Crystal Structure of CYP2B6 in Complex with an Efavirenz Analog

“…Out of the 57 human CYP enzymes, about a dozen are involved in drug metabolism, including enzymes from the CYP1, 2 and 3 families [ 2 ]. Over the past several decades, our laboratory has focused on characterizing the CYP2B subfamily of enzymes in the absence and presence of a wide array of xenobiotics using functional, structural, computational, and biophysical methods [ 3 , 4 , 5 , 6 , 7 ]. The functional differences observed among these enzymes, which include rat CYP2B1, rabbit CYP2B4, human CYP2B6, dog CYP2B11, and woodrat CYP2B35 and 37, have allowed in-depth exploration of structure-function relationships of direct relevance to drug metabolism by other CYP families [ 8 ].…”

Role of cytochrome P450 2B sequence variation and gene copy number in facilitating dietary specialization in mammalian herbivores

“…Purified woodrat CYP2B enzymes display high affinities towards specific terpenes found in juniper, suggesting the importance of these enzymes in the metabolism of juniper PSMs (Wilderman, Shah, Jang, Stout, & Halpert, 2013;Wilderman et al, 2014). Finally, woodrats have multiple gene copies of CYP2B that encode enzymes with varying substrate specificities (Malenke et al, 2012;Shah et al, 2016).…”