Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms

Yoshimoto, F. K.; Auchus, Richard J.

doi:10.1016/j.jsbmb.2015.10.005

Cited by 9 publications

(7 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CPR transfers two electrons sequentially to CYPs during the catalytic monooxygenation cycle. The second ET step is rate-determining for most bacterial CYPs 58 , whereas for eukaryotic CYPs, different rate-determining steps—formation of the membrane-bound CYP–CPR complex 59 , product release 36 , or the first 60 or second ET 61 —have been reported. The first ET to mammalian CYPs from CPR occurs at ~2–10 s −1 62 , 63 and the second ET is usually within this range or slower 45 , 62 , 64 .…”

Section: Resultsmentioning

confidence: 99%

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

2021

View full text Add to dashboard Cite

Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

show abstract

Section: Resultsmentioning

confidence: 99%

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

2021

View full text Add to dashboard Cite

show abstract

“…[155] Applications of stopped-flow methods to kinetic measurements, specifically for cytochromes P450, were recently reviewed. [156]…”

Section: Substrate Bindingmentioning

confidence: 99%

Spectroscopic studies of the cytochrome P450 reaction mechanisms

Mak

Denisov

2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

View full text Add to dashboard Cite

The cytochrome P450 monooxygenases (P450s) are thiolate heme proteins that can, often under physiological conditions, catalyze many distinct oxidative transformations on a wide variety of molecules, including relatively simple alkanes or fatty acids, as well as more complex compounds such as steroids and exogenous pollutants. They perform such impressive chemistry utilizing a sophisticated catalytic cycle that involves a series of consecutive chemical transformations of heme prosthetic group. Each of these steps provides a unique spectral signature that reflects changes in oxidation or spin states, deformation of the porphyrin ring or alteration of dioxygen moieties. For a long time, the focus of cytochrome P450 research was to understand the underlying reaction mechanism of each enzymatic step, with the biggest challenge being identification and characterization of the powerful oxidizing intermediates. Spectroscopic methods, such as electronic absorption (UV-Vis), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electron nuclear double resonance (ENDOR), Mössbauer, X-ray absorption (XAS), and resonance Raman (rR), have been useful tools in providing multifaceted and detailed mechanistic insights into the biophysics and biochemistry of these fascinating enzymes. The combination of spectroscopic techniques with novel approaches, such as cryoreduction and Nanodisc technology, allowed for generation, trapping and characterizing long sought transient intermediates, a task that has been difficult to achieve using other methods. Results obtained from the UV-Vis, rR and EPR spectroscopies are the main focus of this review, while the remaining spectroscopic techniques are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

show abstract

“…The second ET step is rate-determining for most bacterial CYPs, 53 whereas for eukaryotic CYPs, different rate-determining steps -formation of the membrane-bound CYP-CPR complex 54 , product release 36 , or the first 55 or second ET 56 -have been reported. The first ET to mammalian CYPs from CPR occurs at ~2-10 s −1 57,58 and the second ET is usually within this range or slower.…”

Section: Simulations Reveal Two Et Pathways and Et Rates Consistent Wmentioning

confidence: 99%

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Mukherjee

Nandekar

Wade

2020

Preprint

View full text Add to dashboard Cite

Cytochrome P450 (CYP) enzymes are heme monooxygenases that require two electrons for their catalytic cycle. For the mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). CYP and CPR each attach to the membrane of the endoplasmic reticulum by a single transmembrane helix with their catalytic domains in the cytosol. No structure of a mammalian CYP-CPR complex has been solved experimentally, hindering a detailed understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the structure and dynamics of the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach, combining Brownian dynamics, coarse-grained and allatom molecular dynamics simulations. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage between the membrane and the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP-CPR ET rates are low compared to those of soluble bacterial CYPs. TOC GRAPHICS

show abstract

Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms

Cited by 9 publications

References 58 publications

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Spectroscopic studies of the cytochrome P450 reaction mechanisms

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

Contact Info

Product

Resources

About