scholarly journals Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency

2015 ◽  
Vol 468 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Yi Jin ◽  
Mo Chen ◽  
Trevor M. Penning ◽  
Walter L. Miller
Keyword(s):  
Electron Transfer ◽  
European Ancestry ◽  
Wild Type ◽  
Transient Kinetics ◽  
Skeletal Malformation ◽  
P450 Oxidoreductase ◽  
Sex Development ◽  
Cytochrome P450 Oxidoreductase ◽  
Flavin Binding ◽  
Human Wild Type

We show that the cytochrome P450 oxidoreductase (POR) A287P variant, which can lead to skeletal malformation and disordered sex development, found in 40% of patients of European ancestry is characterized by deficient flavin binding and impaired electron transfer from NADPH.

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

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.

scholarly journals Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction

Antioxidants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 673
Author(s):  
Masakazu Sugishima ◽  
Junichi Taira ◽  
Tatsuya Sagara ◽  
Ryota Nakao ◽  
Hideaki Sato ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Enzymatic Activity ◽  
Heme Oxygenase ◽  
Analytical Ultracentrifugation ◽  
Dynamic Equilibrium ◽  
P450 Oxidoreductase ◽  
Nadph Cytochrome ◽  
Redox Partner ◽  
Cytochrome P450 Oxidoreductase

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1.

scholarly journals Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase

2018 ◽  
Vol 129 (4) ◽  
pp. 756-768 ◽  
Author(s):  
Pan-Fen Wang ◽  
Alicia Neiner ◽  
Evan D. Kharasch
Keyword(s):  
Cytochrome P450 ◽  
Genetic Variants ◽  
Cytochrome B5 ◽  
Intrinsic Clearance ◽  
Cell System ◽  
Therapeutic Effects ◽  
Wild Type ◽  
P450 Oxidoreductase ◽  
Cytochrome P450 Oxidoreductase

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.

scholarly journals 46,XX DSD and Antley-Bixler syndrome due to novel mutations in the cytochrome P450 oxidoreductase gene

2012 ◽  
Vol 56 (8) ◽  
pp. 578-585 ◽  
Author(s):  
Guilherme Guaragna-Filho ◽  
Carla Cristina Telles de Sousa Castro ◽  
Rodrigo Ribeiro De Carvalho ◽  
Fernanda Borchers Coeli ◽  
Lúcio Fábio Caldas Ferraz ◽  
...  
Keyword(s):  
Disorders Of Sex Development ◽  
First Year ◽  
The Novel ◽  
Hypergonadotropic Hypogonadism ◽  
Steroidogenic Enzyme ◽  
P450 Oxidoreductase ◽  
Sex Development ◽  
Skeletal Malformations ◽  
Cytochrome P450 Oxidoreductase ◽  
First Year Of Life

Deficiency of the enzyme P450 oxidoreductase is a rare form of congenital adrenal hyperplasia with characteristics of combined and partial impairments in steroidogenic enzyme activities, as P450 oxidoreductase transfers electrons to CYP21A2, CYP17A1, and CYP19A1. It results in disorders of sex development and skeletal malformations similar to Antley-Bixley syndrome. We report the case of a 9-year-old girl who was born with virilized genitalia (Prader stage V), absence of palpable gonads, 46,XX karyotype, and hypergonadotropic hypogonadism. During the first year of life, ovarian cyst, partial adrenal insufficiency, and osteoarticular changes, such as mild craniosynostosis, carpal and tarsal synostosis, and limited forearm pronosupination were observed. Her mother presented severe virilization during pregnancy. The molecular analysis of P450 oxidoreductase gene revealed compound heterozygosis for the nonsense p.Arg223*, and the novel missense p.Met408Lys, inherited from the father and the mother, respectively. Arq Bras Endocrinol Metab. 2012;56(8):578-85

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

2020 ◽  
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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