Interaction modes of microsomal cytochrome p450s with its reductase and the role of substrate binding

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR’s degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP’s substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound.publishersversionpublishe

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

[This corrects the article DOI: 10.3389/fphar.2020.00299.].publishersversionpublishe

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism

Funding Information: F.E. and M.K.: UID/BIM/0009/2020 of the Portuguese Fundação para a Ciência e a Tecnologia (FCT) and HLTH-2022-STAYHLTH-02/grant agreement 101095679 of the European Horizon´s research and innovation program. Publisher Copyright: © 2023 by the authors.A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD–CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.publishersversionpublishe

Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments

International audiencePhytoene desaturases catalyse from two to six desaturation reactions on phytoene, generating a large diversity of molecules that can then be cyclised and produce, depending on the organism, many different carotenoids. We constructed a phylogenetic tree of a subset of phytoene desaturases from the CrtI family for which functional data was available. We expressed in a bacterial system eight codon optimized CrtI enzymes from different clades. Analysis of the phytoene desaturation reactions on crude extracts showed that three CrtI enzymes can catalyse up to six desaturations, forming tetradehydrolycopene. Kinetic data generated using a subset of five purified enzymes demonstrate the existence of characteristic patterns of desaturated molecules associated with various CrtI clades. The kinetic data was also analysed using a classical Michaelis-Menten kinetic model, showing that variations in the reaction rates and binding constants could explain the various carotene patterns observed. Competition between lycopene cyclase and the phytoene desaturases modified the distribution between carotene intermediates when expressed in yeast in the context of the full β-carotene production pathway. Our results demonstrate that the desaturation patterns of carotene molecules in various biological environments cannot be fully inferred from phytoene desaturases classification but is governed both by evolutionary-linked variations in the desaturation rates and competition between desaturation and cyclisation steps

Conséquences de l'échange de domaines évolutivement éloignés sur l'activité et la géométrie de la nadph-cytochrome P450 réductase

Les cytochromes P450 (P450) et la NADPH-cytochrome P450 reductase (CPR) sont les principaux acteurs du métabolisme des xénobiotiques chez les mammifères. La CPR est une protéine multidomaine formée de deux domaines catalytiques comprenant les cofacteurs FAD et FMN, et reliés par un domaine de connexion. Grace aux propriétés rédox de ses deux flavines, la CPR est capable de scinder le flux diélectronique du NADPH en deux transferts monoélectroniques vers les P450. Jusqu alors, la CPR avait toujours cristallisée dans une conformation dite fermée, compatible avec un transfert interne du FAD au FMN, mais dans laquelle le FMN été tellement enfuit au cœur de la protéine qu un transfert externe vers les P450 était inenvisageable. Le projet de cette thèse consistait à construire et analyser les caractéristiques structurales et biochimiques de CPR chimères constituées de domaines issues des CPR humaine et de levure. Ces chimères sont toujours fonctionnelles vis-à-vis d accepteurs artificiels et naturels. De plus, la structure cristallographique de l une d elle a pu être déterminée à 2,5 Å de résolution et dans une nouvelle conformation dites ouverte. Celle-ci prouve le caractère dynamique de cette protéine multidomaine, et pourrait correspondre à la conformation de la CPR au sein du complexe bimoléculaire CPR-P450 lors du transfert externe entre le FMN et le cytochrome. Les facteurs pouvant influencer ou dicter ces changements conformationnels ont été appréhendés grâce à des analyses d enzymologie, de cinétiques rapides, de potentiométrie ou encore de dénaturation.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Sequence Mapping of Combinatorial Libraries on Macro- and Microarrays Bioinformatic Treatment of Data

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High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation

Producing Chimeric Genes by CLERY In Vitro and In Vivo Recombination

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Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes

Background: A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. [br/][br/]Methods: Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. [br/][br/]Results: The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. [br/][br/]Conclusions: Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. General significance: Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active sit

Le cytochrome b5 humain comme modèle dans les mécanismes d'interactions pour la formation du complexe protéine-protéine dans les réactions de transport d'électrons du réticulum endoplasmique

Les mécanismes de reconnaissance qui gouvernent les réactions de transfert d'électrons entre partenaires redox du réticulum endoplasmique ne sont pas complètement décrits. Nous avons étudié ce système en utilisant comme modèle le cytochrome b5 (cytb5), membre d'un complexe formé par le cytochrome P450 3A4 (CYP3A4) et la NADPH-cytochrome P450 réductase. Ces protéines sont constituées par un domaine hydrosoluble qui contient le centre redox et un domaine hydrophobe qui ancre la protéine à la membrane. Dans le cas du cytb5, ces deux segments sont reliés par un segment dit de jonction. Notre modèle d'étude se base sur les propriétés différentes de reconnaissance entre deux cytb5 de différentes espèces (humain et levure) et le CYP3A4 humain. Les études biochimiques sur les propriétés des protéines mosaïques montrent qu'une part de la reconnaissance entre cytb5 et CYP3A4 est modulée par le segment de jonction et une partie du domaine membranaire. Nous présentons également une analyse structurale et dynamique par RMN du liquide de la partie hydrosoluble du cytb5 humain, ainsi qu'une approche structurale du domaine membranaire de cette protéine.Protein recognition mechanisms which control electron transfers between redox partners in the endoplasmic reticulum are not completely understood. We have studied this system using, as a model, the cytochrome b5 (cytb5), member of a multi-protein complex formed by the P450 3A4 (CYP3A4) and NADPH-cytochrome P450 réductase. These proteins consist of a water-soluble domain that contains the redox center and a hydrophobic domain which anchors protein to the membrane. In the case of cytb5, these two segments are connected by a junction segment. Our study is based on the different recognition properties of two cytb5 of various species (human and yeast) with the human CYP3A4. The biochemical studies on the properties of mosaic proteins show that a part of the recognition between cytb5 and CYP3A4 is modulated by the junction segment and part of the membrane domain. We also present a structural and dynamic analysis by liquid NMR of the hydrophylic part of the human cytb5, as well as a starting approach of the structural properties of the membrane domain of this protein.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF