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

Francisco Esteves, Cristina M.M. Almeida, Sofia Silva, Inês Saldanha, Philippe Urban, José Rueff, Denis Pompon, Gilles Truan, Michel Kranendonk

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)
8 Downloads (Pure)

Abstract

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.

Original languageEnglish
Article number1083
JournalBiomolecules
Volume13
Issue number7
DOIs
Publication statusPublished - Jul 2023

Keywords

  • caffeine metabolism
  • cytochrome P450
  • enzyme mechanism
  • enzyme mutation
  • protein dynamics
  • reductase

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