title: Kinetic analysis of cytochrome P450 reductase from Artemisia annua reveals accelerated rates of NADH-dependent flavin reduction.
abstract: Cytochrome P450 reductase from Artemisia annua (aaCPR) is a diflavin enzyme that has been employed for the microbial synthesis of artemisinic acid (a semi-synthetic precursor of the anti-malarial drug, artemisinin) based on its ability to transfer electrons to the cytochrome P450 monooxygenase, CYP71AV1. We have isolated recombinant aaCPR (with the N-terminal transmembrane motif removed) from Escherichia coli and compared its kinetic and thermodynamic properties with other CPR orthologues, most notably human CPR. The FAD and FMN redox potentials and the macroscopic kinetic constants associated with cytochrome c(3+) reduction for aaCPR are comparable to that of other CPR orthologues, with the exception that the apparent binding affinity for the oxidized coenzyme is ~ 30-fold weaker compared to human CPR. CPR from A. annua shows a 3.5-fold increase in uncoupled NADPH oxidation compared to human CPR and a strong preference (85 100-fold) for NADPH over NADH. Strikingly, reduction of the enzyme by the first and second equivalent of NADPH is much faster in aaCPR, with rates of > 500 and 17 s(-1) at 6 °C. We also optically detect a charge-transfer species that rapidly forms in < 3 ms and then persists during the reductive half reaction. Additional stopped-flow kinetic studies with NADH and (R)-[4-(2) H]NADPH suggest that the accelerated rate of flavin reduction is attributed to the relatively weak binding affinity of aaCPR for NADP(+) .
authors.affiliation: Department of Chemistry, University of British Columbia, Kelowna, BC, Canada.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Simtchouk
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Eng
authors.forename: Carla E
authors.initials: CE
authors.lastname: Meints
authors.forename: Caitlyn
authors.initials: C
authors.lastname: Makins
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Wolthers
mesh_terms: D000595:Amino Acid Sequence; D031172:Artemisia annua / Q000201:enzymology*; D037621:Artemisinins / Q000378:metabolism; D003001:Cloning, Molecular; D004579:Electron Transport; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D037621:Artemisinins; D005415:Flavins; D009243:NAD; C047721:artemisic acid; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Artemisia annua; cytochrome P450 reductase; flavin; potentiometry; stopped-flow spectroscopy