Functional characterization of eight human CYP1A2 variants

Palma, Bernardo Brito; Sousa, Marta Silva E; Urban, Philippe; Rueff, José; Kranendonk, Michel

doi:10.1097/fpc.0b013e32835c2ddf

Cited by 24 publications

(37 citation statements)

References 57 publications

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“…Cultures of CPR wt /CYP and CPR null /CYP were used as controls in all experiments. Control strains were obtained for each CYP (1A2, 2A6, or 3A4): one expressing CPR wt , through expression plasmid pLCM_POR wt , or with the mock plasmid pLCM (lacking POR cDNA), each combined with the respective human CYP expression plasmid (pCW_CYP) (Kranendonk et al, 1998;Palma et al, 2013). Clones demonstrating reaction velocities above 110%, i.e., an increase above two times background variation (5%), were selected.…”

Section: High-throughput Screening Of Cpr-fmn-domain Mutant-librariesmentioning

confidence: 99%

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

et al. 2020

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NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b 5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches-formerly indicated to be involved in CPR:CYP interactions-or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

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Section: High-throughput Screening Of Cpr-fmn-domain Mutant-librariesmentioning

confidence: 99%

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

et al. 2020

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“…Effects of b 5 are however variable, stimulating, inhibiting, or not affecting metabolism, depending on the P450 isoform, substrate, and experimental conditions (Schenkman and Jansson, 2003;Finn et al, 2008;Im and Waskell, 2011). Recently this variability was shown for the first time to extend to P450 variants, with some CYP1A2 mutants quite affected by b 5 (Palma et al, 2013). On the basis of several kinetic, mutagenesis, and NMR binding studies of wild-type rabbit CYP2B4, Waskell et al proposed a model for an electron transfer complex between the acidic convex surface of b 5 and the concave basic proximal surface of CYP2B4 (Im and Waskell, 2011;Ahuja et al, 2013).…”

Section: Downloaded Frommentioning

confidence: 99%

“…The mutated residues in CYP2B6.6 (Gln172His, Lys262Arg), however, do not correspond to the b 5 binding site identified on CYP2B4. With CYP1A2, the variant Gly299Ser (CYP1A2.13) had the most altered b 5 response compared with wild-type, with a lesser influence of the variants Thr83Met (CYP1A2.9), Ile386Phe (CYP1A2.4), and Cys406Tyr (CYP1A2.5) (Palma et al, 2013). Gly299Ser was reported to be on the surface of the heme domain near the interaction site with b 5 , and close to the CYP1A2 C-helix, which is also thought to be important in interaction with b 5 (Palma et al, 2013).…”

Section: Downloaded Frommentioning

confidence: 99%

“…With CYP1A2, the variant Gly299Ser (CYP1A2.13) had the most altered b 5 response compared with wild-type, with a lesser influence of the variants Thr83Met (CYP1A2.9), Ile386Phe (CYP1A2.4), and Cys406Tyr (CYP1A2.5) (Palma et al, 2013). Gly299Ser was reported to be on the surface of the heme domain near the interaction site with b 5 , and close to the CYP1A2 C-helix, which is also thought to be important in interaction with b 5 (Palma et al, 2013). On the basis of sequence alignment of CYP2B6 with CYP1A2, Thr83, Gly299, Ile386, and Cys406 of CYP1A2 correspond to CYP2B6 Arg73, His280, Val367, and Ile387.…”

Section: Downloaded Frommentioning

confidence: 99%

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Differences in Methadone Metabolism by CYP2B6 Variants

2015

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Methadone is a long-acting opioid with considerable unexplained interindividual variability in clearance. Cytochrome P450 2B6 (CYP2B6) mediates clinical methadone clearance and metabolic inactivation via N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Retrospective studies suggest that individuals with the CYP2B6*6 allelic variant have higher methadone plasma concentrations. Catalytic activities of CYP2B6 variants are highly substrate-and expression-system dependent. This investigation evaluated methadone N-demethylation by expressed human CYP2B6 allelic variants in an insect cell coexpression system containing P450 reductase. Additionally, the influence of coexpressing cytochrome b 5 , whose role in metabolism can be inhibitory or stimulatory depending on the P450 isoform and substrate, on methadone metabolism, was evaluated. EDDP formation from therapeutic (0.25-1 mM) R-and S-methadone concentrations was CYP2B6.4 ‡ CYP2B6.1 ‡ CYP2B6.5 >> CYP2B6.9 CYP2B6.6, and undetectable from CYP2B6.18. Coexpression of b 5 had small and variant-specific effects at therapeutic methadone concentrations but at higher concentrations stimulated EDDP formation by CYP2B6.1, CYP2B6.4, CYP2B6.5, and CYP2B6.9 but not CYP2B6.6. In vitro intrinsic clearances were generally CYP2B6.4 ‡ CYP2B6.1 > CYP2B6.5 > CYP2B6.9 ‡ CYP2B6.6. Stereoselective methadone metabolism (S>R) was maintained with all CYP2B6 variants. These results show that methadone N-demethylation by CYP2B6.4 is greater compared with CYP2B6.1, whereas CYP2B6.9 and CYP2B6.6 (which both contain the 516G>T, Q172H polymorphism), are catalytically deficient. The presence or absence of b 5 in expression systems may explain previously reported disparate catalytic activities of CYP2B6 variants for specific substrates. Differences in methadone metabolism by CYP2B6 allelic variants provide a mechanistic understanding of pharmacogenetic variability in clinical methadone metabolism and clearance.

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“…Expression of the full-length membrane bound CPR mutants was obtained in BTC bacteria and membrane fractions of the different strains were prepared and characterized for protein content as described previously Palma et al, 2013). CPR content of membrane fractions was quantified by immunodetection against a standard curve of purified human, full-length WT CPR, using polyclonal rabbit anti-CPR primary antibody and biotin-goat anti-rabbit antibody in combination with the fluorescent streptavidin conjugate (WesternDot 625 Western Blot Kit; Invitrogen).…”

Section: Membrane-bound Forms Of Cprmentioning

confidence: 99%

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

Pandey¹,

Henderson²,

Ishii³

et al. 2018

Frontiers Research Topics

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Functional characterization of eight human CYP1A2 variants

Abstract: Results indicate the ability of b5 to affect CYP1A2 variants to behave more like the wild-type variant, attenuating detrimental effects of structural mutations of these variants, seemingly through extensive allosteric effects.

Cited by 24 publications

References 57 publications

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

Differences in Methadone Metabolism by CYP2B6 Variants

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

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