Role of LYS271 and LYS279 Residues in the Interaction of Cytochrome P4501A1 with NADPH–Cytochrome P450 Reductase

Cvrk, Tomas; Strobel, Henry W.

doi:10.1006/abbi.2000.2174

Cited by 13 publications

(7 citation statements)

References 35 publications

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“…Previous studies revealed that basic amino acids on the proximal surface near the heme were involved in the binding of CYP2B4 and 1A1 with b 5 or CPR (Bridges et al, 1998;Cvrk and Strobel, 2001). Our results indicate that each redox partner affects the binding of the other partner with CYP3A4, which may be due to the overlapping binding sites.…”

Section: Discussionmentioning

confidence: 51%

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

Yamaguchi¹,

Khan²,

He³

et al. 2004

Drug Metab Dispos

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Section: Discussionmentioning

confidence: 51%

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

Yamaguchi¹,

Khan²,

He³

et al. 2004

Drug Metab Dispos

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“…Earlier mutagenesis studies by Cvrk and Strobel indicated that these residues were involved in substrate binding inside the active site of CYP1A1 (43,44). Corresponding residues in BM-3, CYP2C9, CYP2D6, and CYP2E1 have also been suggested to have a role in substrate specificity (21,(45)(46)(47).…”

Section: Resultsmentioning

confidence: 94%

Ensemble Modeling of Substrate Binding to Cytochromes P450: Analysis of Catalytic Differences between CYP1A Orthologs^,

et al. 2007

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A novel application of modeling and docking approaches involving ensembles of homology models is used to understand structural bases underlying subtle catalytic differences between related cytochromes P450 (CYPs). Mammalian CYP1A1s and fish CYP1As are orthologous enzymes with similar substrate preferences. With some substrates (3,3',4,4'-tetrachlorobiphenyl, TCB) oxidation rates differ by orders of magnitude, while others (e.g., benzo[a]pyrene; B[a]P) are oxidized at similar rates but with somewhat differing regiospecificity. These two environmental chemical substrates (TCB and B[a]P) as well as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were docked to multiple models of rat, human, scup, and/or killifish CYP1As, based on multiple templates, retaining multiple poses from each model, giving ensembles of docked poses for each species. With TCB, more poses were observed closer to the heme in ensembles of rat or human CYP1A1 than of killifish CYP1A. Analysis of interacting residues suggested that differences in TCB pose distributions are due primarily to Leu387 and Val230 in killifish CYP1A. In silico mutations L387V and V230G enabled TCB to dock closer to the heme in killifish CYP1A. Mutating additional interacting residues (Ala127, Thr233, Asn317, and Tyr386) of killifish CYP1A to the corresponding residues of human CYP1A1 resulted in TCB pose distributions nearly identical with those of human CYP1A1. Docking of TCDD to sets of consensus models of killifish, rat, and human CYP1As showed species differences similar to those with TCB, but with further structural constraints possibly contributing to slower oxidation of TCDD. Docking B[a]P to sets of consensus models of the human and fish CYP1As yielded frequencies of substrate orientations correlating with known regiospecificities for metabolism of B[a]P by these enzymes. The results demonstrate the utility of this ensemble modeling method, which can account for uncertainty inherent in homology modeling and docking by producing statistical distributions of ligand positions.

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“…There is experimental evidence supporting interactions between the NADP domain and the CYP globular domain for the CYP 1A1-CPR complex. A strong inhibition (>80%) of the CPR-supported metabolism of 7-ethoxycoumarin and ethoxyresorufin was observed in K271I and K279I single-point mutants of rat CYP 1A1, accompanied by a reduction by a factor of about 2 and 9, respectively, of the Michaelis constant for the reductase (wild-type K m = 5.1 pM) 53 . Based on a combination of in vitro mutagenesis, in vivo screening, and spectral analysis, it was confirmed that K268 and R275 of rat CYP 1A1 are important for CPR binding 54,55 .…”

Section: Resultsmentioning

confidence: 99%

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

2021

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Cytochrome 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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Role of LYS271 and LYS279 Residues in the Interaction of Cytochrome P4501A1 with NADPH–Cytochrome P450 Reductase

Cited by 13 publications

References 35 publications

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

Ensemble Modeling of Substrate Binding to Cytochromes P450: Analysis of Catalytic Differences between CYP1A Orthologs^,

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

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Role of LYS271 and LYS279 Residues in the Interaction of Cytochrome P4501A1 with NADPH–Cytochrome P450 Reductase

Cited by 13 publications

References 35 publications

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB5AND OF SITE-DIRECTED MUTAGENESIS

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB5AND OF SITE-DIRECTED MUTAGENESIS

Ensemble Modeling of Substrate Binding to Cytochromes P450: Analysis of Catalytic Differences between CYP1A Orthologs,

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

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TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

TOPOLOGICAL CHANGES IN THE CYP3A4 ACTIVE SITE PROBED WITH PHENYLDIAZENE: EFFECT OF INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE AND CYTOCHROMEB₅AND OF SITE-DIRECTED MUTAGENESIS

Ensemble Modeling of Substrate Binding to Cytochromes P450: Analysis of Catalytic Differences between CYP1A Orthologs^,