A Homodimer Model Can Resolve the Conundrum as to How Cytochrome P450 Oxidoreductase and Cytochrome b5 Compete for the Same Binding Site on Cytochrome P450c17

Abstract Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. Methods CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography–mass spectrometry. Michaelis–Menten kinetic parameters were determined. Results For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Conclusions Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

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Differential Contributions of NADPH-Cytochrome P450 Oxidoreductase FAD Binding Site Residues to Flavin Binding and Catalysis

Journal of Biological Chemistry ◽

10.1074/jbc.m008380200 ◽

2000 ◽

Vol 275 (52) ◽

pp. 41087-41091 ◽

Cited By ~ 30

Author(s):

Anna L. Shen ◽

Charles B. Kasper

Keyword(s):

Cytochrome P450 ◽

Binding Site ◽

P450 Oxidoreductase ◽

Nadph Cytochrome ◽

Cytochrome P450 Oxidoreductase ◽

Flavin Binding ◽

Fad Binding

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An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Communications Biology ◽

10.1038/s42003-020-01568-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Goutam Mukherjee ◽

Prajwal P. Nandekar ◽

Rebecca C. Wade

Keyword(s):

Electron Transfer ◽

Cytochrome P450 ◽

Drug Targets ◽

Catalytic Domain ◽

Cytochrome P450 1A1 ◽

P450 Oxidoreductase ◽

Distal Side ◽

Cytochrome P450 Oxidoreductase ◽

Membrane Bound ◽

Transient Interactions

AbstractCytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

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