scholarly journals Substrate mediated redox partner selectivity of cytochrome P450

2018 ◽  
Vol 54 (45) ◽  
pp. 5780-5783 ◽  
Author(s):  
Katherine A. Gentry ◽  
Meng Zhang ◽  
Sang-Choul Im ◽  
Lucy Waskell ◽  
Ayyalusamy Ramamoorthy
Keyword(s):  
Cytochrome P450 ◽  
Hydrophobic Drugs ◽  
Redox Partner ◽  
Redox Partners

Investigating the interplay between cytochrome-P450 and its redox partners (CPR and cytochrome-b5) is vital for understanding the metabolism of most hydrophobic drugs.

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.

Comparative Analysis of Three Pairs of Surrogate Redox Partners for in Vitro Activity Reconstitution of Class I Cytochrome P450 Enzymes

2021 ◽  
Author(s):  
Xiaohui Liu ◽  
Fengwei Li ◽  
Tianjian Sun ◽  
Jiawei Guo ◽  
Xingwang Zhang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Experimental Studies ◽  
Class I ◽  
Cytochrome P450 Enzymes ◽  
Catalytic Systems ◽  
Redox Partner ◽  
Redox Partners ◽  
Lowering Drug ◽  
Electron Transfer Properties

Abstract Cytochrome P450 enzymes (P450s) are highly attractive biocatalysts due to their versatile catalytic activities. A vast majority of P450s require redox partner (RP) proteins to sequentially transfer two electrons for O2 activation and substrate oxidation. However, little information is available on cognate RPs for P450s, which greatly limits P450 function exploration and practical application. Thus, the stategy of building various hybrid P450 catalytic systems with surrogate RPs has often adopted to engineer P450 biocatalysts for different purposes. In this study, we comprehensively compare three pairs of frequently-used surrogate redox partners SelFdx1499/SelFdR0978, Adx/AdR and Pdx/PdR and in terms of their electron transfer properties. The three selected bacterial Class I P450s to accept electrons from RPs include PikC, P450sca-2 and CYP-sb21, which are responsible for production of macrolide antibiotics, the cholesterol-lowering drug pravastatin, and a hair-growth-stimulating agent. Both experimental studies and structural analysis show that SelFdx1499/SelFdR0978 is the most promising RP compared to Adx/AdR and Pdx/PdR. The results provide insights into the domination for P450-redox partner interactions in modulating the catalytic activity of P450s. This study not only produces a more active biocatalyst but also suggests a general chose for a universal reductase which would facilitate engineering of P450 catalyst.

scholarly journals Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450

2018 ◽  
Vol 19 (12) ◽  
pp. 3914 ◽  
Author(s):  
Diana Campelo ◽  
Francisco Esteves ◽  
Bernardo Brito Palma ◽  
Bruno Costa Gomes ◽  
José Rueff ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome B5 ◽  
Cytochrome P450s ◽  
Cytochrome P450 Reductase ◽  
Redox Partner ◽  
Redox Partners ◽  
Cytochrome P450 Oxidoreductase ◽  
Experimental Approaches ◽  
Menten Kinetic

NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

scholarly journals Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

2019 ◽  
Vol 166 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Jan M Klenk ◽  
Max-Philipp Fischer ◽  
Paulina Dubiel ◽  
Mahima Sharma ◽  
Benjamin Rowlinson ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathway ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Cytochrome P450 Monooxygenases ◽  
Dihydroxyphenylacetic Acid ◽  
Meta Position ◽  
Redox Partners

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners

Biochimie ◽  
2019 ◽  
Vol 162 ◽  
pp. 156-166 ◽  
Author(s):  
Evgeniy O. Yablokov ◽  
Tatsiana A. Sushko ◽  
Pavel V. Ershov ◽  
Anna V. Florinskaya ◽  
Oksana V. Gnedenko ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Comparative Analysis ◽  
Large Scale ◽  
Functional Characteristics ◽  
Redox Partners ◽  
Cytochrome P450 Isoforms

scholarly journals Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

2019 ◽  
Vol 103 (9) ◽  
pp. 3597-3614 ◽  
Author(s):  
Sandra Ortega Ugalde ◽  
Maikel Boot ◽  
Jan N. M. Commandeur ◽  
Paul Jennings ◽  
Wilbert Bitter ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
Drug Targets ◽  
Cytochrome P450 Enzymes ◽  
Redox Partners

scholarly journals Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding

2011 ◽  
Vol 286 (18) ◽  
pp. 16246-16260 ◽  
Author(s):  
Chuanwu Xia ◽  
Djemel Hamdane ◽  
Anna L. Shen ◽  
Vivian Choi ◽  
Charles B. Kasper ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Disulfide Bond ◽  
Conformational Changes ◽  
Wild Type ◽  
Disulfide Linkage ◽  
Nadph Cytochrome ◽  
Redox Partners ◽  
Domain Movement ◽  
Flavin Domain

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

Evolution of the soluble nitrate reductase: defining the monomeric periplasmic nitrate reductase subgroup

2006 ◽  
Vol 34 (1) ◽  
pp. 122-126 ◽  
Author(s):  
B.J.N. Jepson ◽  
A. Marietou ◽  
S. Mohan ◽  
J.A. Cole ◽  
C.S. Butler ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Structural Alignment ◽  
Bioinformatic Analysis ◽  
Catalytic Subunit ◽  
Surface Polarity ◽  
Redox Partner ◽  
Redox Partners ◽  
Membrane Bound ◽  
And Function ◽  
Nitrate Reductases

Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.

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