Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase:cytochrome P-450 interaction.

Peterson, Julian A.; Ebel, Richard E.; O'Keeffe, David H.; Matsubara, Takashi; Estabrook, Ronald W.

doi:10.1016/s0021-9258(17)33349-5

Cited by 242 publications

(46 citation statements)

References 41 publications

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“…In humans, 57 cytochrome P450 isoforms with defined catalytic specificity are responsible for xenobiotic metabolism and biosynthesis, , which require fine-tuned dynamic regulation . CPR needs to provide electrons to each of the different P450 present, and it has been speculated that CPR conformational plasticity is essential for both electron transfer and the formation of a functional complex with P450. , The stoichiometric disproportion between CPR and cytochromes P450s has prompted several hypotheses, including quaternary organization of P450, which are sustained by kinetic results obtained in ensemble experiments. Herein we rely on direct observation through single-protein tracking of individual CPR and P450 interacting with a planar supported bilayer that mimic the chemical composition of the ER.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, CPR is also the main electron transferase for other ER membrane enzymes, including squalene monooxygenase and heme oxygenase. , In order to explain the efficiency of the electron transfer between CPR and several P450 units in a membrane environment, two hypotheses have arisen: (1) a diffusion-controlled model, in which the frequency of protein–protein interactions is controlled by the lateral diffusion of the protein(s) in the membrane, and (2) a more static quaternary organization of the proteins as hetero (CPR–P450) or homo (P450–P450) oligomers (collectively known as “protein clusters”). Estabrook suggested the cluster hypothesis in the 1970s, , and it was subsequently adopted by several research groups. , However, a few papers in the same period came to the conclusions that the observed kinetics can also be explained by assuming lateral diffusion through the ER. , …”

Section: Introductionmentioning

confidence: 99%

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Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

2017

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Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured K values are highly dependent on the redox state of CPR. It is shown that CPR/CYP2C9 complexes have a much higher dissociation constant than CPR/CYP2C9 or CPR/CYP2C9 complexes, and a model is presented to account for these results. An Arrhenius analysis of diffusion constants was also carried out, demonstrating that the reduced forms of CPR and CYP2C9 interact differently with the biomimetic ER and may, in addition to protein conformational changes, contribute to the observed NADPH-dependent shift in K. Finally, it is also shown that the CPR/CYP2C9 affinity depends on the nature of the ligand, being higher when a substrate is bound, compared to an inhibitor.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

2017

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“…At the endoplasmic reticulum (ER) surface, the CPR is stoichiometrically disfavored with respect to its many distinct CYP450 partners (ratio is 1:5−20). Hence, because CPR/ CYP450 complexes are functional in a 1:1 ratio, the CPR protein must overcome this stoichiometric imbalance to efficiently perform its function, 4 promptly dissociating from its redox partner after ET has occurred.…”

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confidence: 99%

All-Atom Simulations Disclose How Cytochrome Reductase Reshapes the Substrate Access/Egress Routes of Its Partner CYP450s

Ritacco

Saltalamacchia

Spinello

et al. 2020

J. Phys. Chem. Lett.

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Cytochromes P450 enzymes (CYP450s) promote the oxidative metabolism of a variety of substrates via the electrons supplied by the cytochrome P450 reductase (CPR) and upon formation of a CPR/CYP450 adduct. In spite of the pivotal regulatory importance of this process, the impact of CPR binding on the functional properties of its partner CYP450 remains elusive. By performing multiple microsecond-long all-atom molecular dynamics simulations of a 520 000-atom model of a CPR/CYP450 adduct embedded in a membrane mimic, we disclose the molecular terms for their interactions, considering the aromatase (HA) enzyme as a proxy of the CYP450 family. Our study strikingly unveils that CPR binding alters HA’s functional motions, bolstering a change in the shape and type of the channels traveled by substrates/products during their access/egress to/from the enzyme’s active site. Our outcomes unprecedentedly contribute to extricate the many entangled facets of the CYP450 metabolon, redrafting its intricate panorama from an atomic-level perspective.

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“…CYP-CPR complexes are known to function at CYP:CPR ratios ranging from 5:1 to 20:1 in the ER. 15,19,30,31 Our copy number modulation test with KO75, KAH82 and SrCPR1 showed that two copies of KO75 was a requirement for an increase in steviol production pointing to a limitation at the KO step. However, addition of a copy of KO (KO:KAH:CPR ratio of 2:1:1) did not require addition of an extra copy of SrCPR1 to yield higher steviol than a 1:1:1 ratio, suggesting that the KO:CPR ratio is suboptimal at 1:1.…”

Section: ■ Discussionmentioning

confidence: 97%

“…Microsomal CYP monooxygenases are membrane-associated hemoproteins whose activity depend on the transfer of two electrons catalyzed by NADPH-dependent cytochrome P450 reductases (CPR), which are also membrane-bound. , In the endoplasmic reticulum (ER) of plants, CYP and CPR function as a complex held together by protein–protein interactions, where the CPR is the node around which CYP subunits and other associated proteins organize themselves. − Optimization of engineered CYP activity for recombinant production of marketable bioproducts is notoriously difficult . Benchmark work involving CYP optimization is demonstrated in the artemisinin work by Paddon et al CYPs can also function in a system with microsomal cytochrome b5 (CB5) membrane-bound hemoproteins, which require a NADH-dependent CB5 reductase (CBR) flavoprotein. , The recent watershed work by Laursen et al on the biosynthesis of the cyanogenic glucoside dhurrin in sorghum demonstrated copurification of CYPs with CPR, but also CB5 and CBR were enriched in the purified sample …”

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confidence: 99%

A Combinatorial Approach To Study Cytochrome P450 Enzymes forDe NovoProduction of Steviol Glucosides in Baker’s Yeast

et al. 2018

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Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPHdependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

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Temperature dependence of cytochrome P-450 reduction. A model for NADPH-cytochrome P-450 reductase:cytochrome P-450 interaction.

Cited by 242 publications

References 41 publications

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study

All-Atom Simulations Disclose How Cytochrome Reductase Reshapes the Substrate Access/Egress Routes of Its Partner CYP450s

A Combinatorial Approach To Study Cytochrome P450 Enzymes forDe NovoProduction of Steviol Glucosides in Baker’s Yeast

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