scholarly journals Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

2017 ◽  
Vol 139 (37) ◽  
pp. 13193-13199 ◽  
Author(s):  
Dipanwita Batabyal ◽  
Logan S. Richards ◽  
Thomas L. Poulos
Keyword(s):  
Cytochrome P450 ◽  
Conformational Dynamics ◽  
Redox Partner

scholarly journals Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition

2020 ◽  
Vol 295 (6) ◽  
pp. 1637-1645 ◽  
Author(s):  
Min Su ◽  
Sumita Chakraborty ◽  
Yoichi Osawa ◽  
Haoming Zhang
Keyword(s):  
Cytochrome P450 ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Full Length ◽  
The Other ◽  
Redox Partner ◽  
Highly Active ◽  
Heme Domain ◽  
Fatty Acid Hydroxylation ◽  
Cytochrome P450 Family

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min−1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.

scholarly journals A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Catalysts ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1083 ◽  
Author(s):  
Kim-Thoa Nguyen ◽  
Ngọc-Lan Nguyen ◽  
Nguyen Van Tung ◽  
Huy Hoang Nguyen ◽  
Mohammed Milhim ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Retinoic Acid ◽  
Amino Acid Sequence Identity ◽  
Hot Spring ◽  
Cytochromes P450 ◽  
Reading Frame ◽  
Redox Partner ◽  
Redox Partners ◽  
Identification And Characterization ◽  
Low Activity

Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.

scholarly journals Conformational Dynamics of Substrate in the Active Site of Cytochrome P450 BM-3/NPG Complex:  Insights from NMR Order Parameters

2007 ◽  
Vol 129 (3) ◽  
pp. 474-475 ◽  
Author(s):  
Krishna Pratap Ravindranathan ◽  
Emilio Gallicchio ◽  
Ann E. McDermott ◽  
Ronald M. Levy
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Conformational Dynamics ◽  
Order Parameters

scholarly journals Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H

2019 ◽  
Author(s):  
Shaheena Parween ◽  
Giovanna DiNardo ◽  
Francesca Baj ◽  
Chao Zhang ◽  
Gianfranco Gilardi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Treatment Options ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Therapeutic Interventions ◽  
Diagnostic Tools ◽  
Variant Form ◽  
Aromatase Activity ◽  
Allelic Variants ◽  
P450 Oxidoreductase ◽  
Redox Partner

AbstractAromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, result in a reduction of CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284L as well as the POR-P284T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained around 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing towards a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point towards a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284L, P284T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in both the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s

2012 ◽  
Vol 97 (4) ◽  
pp. 1625-1635 ◽  
Author(s):  
Marco Girhard ◽  
Florian Tieves ◽  
Evelyne Weber ◽  
Martha Sophia Smit ◽  
Vlada B. Urlacher
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase ◽  
Redox Partner ◽  
Candida Apicola

scholarly journals Conformational selectivity in cytochrome P450 redox partner interactions

2016 ◽  
Vol 113 (31) ◽  
pp. 8723-8728 ◽  
Author(s):  
Scott A. Hollingsworth ◽  
Dipanwita Batabyal ◽  
Brian D. Nguyen ◽  
Thomas L. Poulos
Keyword(s):  
Cytochrome P450 ◽  
Structural Changes ◽  
Cytochromes P450 ◽  
Substrate Oxidation ◽  
Heme Iron ◽  
Spectral Studies ◽  
Titration Calorimetry ◽  
Nmr Studies ◽  
Redox Partner ◽  
Open Conformation

The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis.

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