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Kinetic analysis of cytochrome P450 reductase from Artemisia annua reveals accelerated rates of NADPH ‐dependent flavin reduction
Author(s) -
Simtchouk Svetlana,
Eng Jordan L.,
Meints Carla E.,
Makins Caitlyn,
Wolthers Kirsten R.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12567
Subject(s) - artemisia annua , flavin group , reductase , monooxygenase , ferredoxin , cytochrome p450 reductase , chemistry , stereochemistry , redox , biochemistry , enzyme , electron transfer , photochemistry , cytochrome p450 , biology , cytochrome c , coenzyme q – cytochrome c reductase , artemisinin , organic chemistry , mitochondrion , plasmodium falciparum , malaria , immunology
Cytochrome P450 reductase from Artemisia annua (aa CPR ) 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, CYP 71 AV 1. We have isolated recombinant aa CPR (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 aa CPR 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 aa CPR , 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 aa CPR for NADP + .