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

José Rueff,Gilles Truan,Sophie Bozonnet,Philippe Urban,Bruno Costa Gomes,Diana Campelo,Michel Kranendonk,Thomas Lautier,Francisco Esteves

doi:10.3389/fphar.2020.00299

Abstract

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 b5 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.

Highlights

NADPH cytochrome P450 oxidoreductase (CPR) is the crucial electron donor of all microsomal cytochrome P450s (CYPs), involved in drug and xenobiotic metabolism, and in other key biochemical processes such as cholesterol and fatty acid homeostasis and steroid hormone biosynthesis (Guengerich, 2015)
Our results seem to suggest that every CYP is interacting with CPR in a specific manner, making use in an isoform specific mode, of docking elements, i.e., a composite of binding motifs of the flavin mononucleotide (FMN)-domain
The capacity of CPR to interact with so many CYPs can be attributed to the presence of different open conformations which are affinity-sampled through the different binding motifs, by which each binding partner can be supplied with electrons

Summary

Introduction

NADPH cytochrome P450 oxidoreductase (CPR) is the crucial electron donor of all microsomal cytochrome P450s (CYPs), involved in drug and xenobiotic metabolism, and in other key biochemical processes such as cholesterol and fatty acid homeostasis and steroid hormone biosynthesis (Guengerich, 2015). The interaction between CPR and microsomal CYPs promotes a two electron transfer (ET) from NADPH through the CPR cofactors FAD (reductase) and FMN (transporter) to the heme iron of CYP (reviewed in, Waskell and Kim, 2015). The highly flexible hinge region which links the FMN-binding domain to the FAD/NADPH-binding domain, is involved in the transition between CPR’s closed/locked (inter-flavin electron transfer) and open/unlocked (electron donation) conformations (Aigrain et al, 2009; Ellis et al, 2009; Hamdane et al, 2009; Sündermann and Oostenbrink, 2013; Frances et al, 2015; Campelo et al, 2017). The hinge region has been suggested to be largely responsible for the formation of diverse multiple open conformations, involved in the formation of productive enzyme complexes, in an apparent CYP isoform dependent manner (Campelo et al, 2018)

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Conclusion