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 Probing protein-protein and protein-substrate interactions in the dynamic membrane-associated ternary complex of Cytochromes P450, b5, and Reductase

2019 ◽  
Author(s):  
Katherine A. Gentry ◽  
G. M. Anantharamaiah ◽  
Ayyalusamy Ramamoorthy
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome B ◽  
Ternary Complex ◽  
Cytochromes P450 ◽  
Dynamic Membrane ◽  
Substrate Interactions ◽  
Protein Substrate ◽  
Redox Partners ◽  
Dynamic Interplay

AbstractCytochrome P450 (cytP450) interacts with two redox partners, cytP450 reductase and cytochrome-b5, to metabolize substrates. Using NMR, we reveal changes in the dynamic interplay when all three proteins are incorporated into lipids nanodiscs in the absence and presence of substrates.

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.

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.

Biotransformation of Benfluron by Rat Hepatic Cytochrome P450. Identification of Individual CYP-Enzymes Involved in Biotransformation of Benfluron, Prospective Antineoplastic Based on Benzo[c]fluorene

2000 ◽  
Vol 65 (8) ◽  
pp. 1374-1386 ◽  
Author(s):  
Kamil Hrubý ◽  
Eva Anzenbacherová ◽  
Pavel Anzenbacher ◽  
Milan Nobilis
Keyword(s):  
Cytochrome P450 ◽  
Drug Interactions ◽  
Enzyme Activities ◽  
Cytochromes P450 ◽  
Antineoplastic Agent ◽  
Marker Enzyme ◽  
Cyp Enzymes ◽  
Cytochrome P450 Isoforms ◽  
Low Activity ◽  
Hepatic Cytochrome

Benfluron, 5-[2-(dimethylamino)ethoxy]-7H-benzo[c]fluoren-7-one hydrochloride, a prospective antineoplastic agent, is metabolised by cytochromes P450 to N-demethyl and 9-hydroxy derivatives. To prove the participation of individual cytochrome P450 isoforms in formation of these metabolites, selective induction of cytochromes P450, inhibition of benfluron biotransformation using inhibitors specific for individual cytochromes P450, and inhibition by benfluron of "marker" enzyme activities characteristic of certain cytochromes P450 were used. N-Demethylbenfluron appears to be formed mainly by the cytochromes P450 of the 3A, 2B and 2C subfamilies with possible participation of the isoform 2E1; 9-hydroxybenfluron is formed with participation of cytochromes P450 belonging to 1A, and most probably to 3A and 2E1 enzymes. The fact that benfluron is in this respect a relatively promiscuous substrate may be an advantage because its metabolism should not be influenced by the absence or low activity of some cytochrome P450 isoforms and by possible drug interactions.

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 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 Probing protein–protein and protein–substrate interactions in the dynamic membrane-associated ternary complex of cytochromes P450, b5, and reductase

2019 ◽  
Vol 55 (89) ◽  
pp. 13422-13425 ◽  
Author(s):  
Katherine A. Gentry ◽  
G. M. Anantharamaiah ◽  
Ayyalusamy Ramamoorthy
Keyword(s):  
Cytochrome P450 ◽  
Ternary Complex ◽  
Cytochromes P450 ◽  
Dynamic Membrane ◽  
Substrate Interactions ◽  
Protein Substrate ◽  
Redox Partners

Cytochrome P450 (cytP450) interacts with two redox partners, cytP450 reductase and cytochrome-b5, to metabolize substrates.

Structural Basis for Effector Control and Redox Partner Recognition in Cytochrome P450

Science ◽  
2013 ◽  
Vol 340 (6137) ◽  
pp. 1227-1230 ◽  
Author(s):  
Sarvind Tripathi ◽  
Huiying Li ◽  
Thomas L. Poulos
Keyword(s):  
Crystal Structure ◽  
Electron Transfer ◽  
Structural Information ◽  
Cytochromes P450 ◽  
Structural Basis ◽  
Proton Coupled Electron Transfer ◽  
Redox Partner ◽  
Open Conformation ◽  
Redox Partners

Cytochromes P450 catalyze a variety of monooxygenase reactions that require electron transfer from redox partners. Although the structure of many P450s and a small handful of redox partners are known, there is very little structural information available on redox complexes, thus leaving a gap in our understanding on the control of P450–redox partner interactions. We have solved the crystal structure of oxidized and reduced P450cam complexed with its redox partner, putidaredoxin (Pdx), to 2.2 and 2.09 angstroms, respectively. It was anticipated that Pdx would favor closed substrate-bound P450cam, which differs substantially from the open conformer, but instead we found that Pdx favors the open state. These new structures indicate that the effector role of Pdx is to shift P450cam toward the open conformation, which enables the establishment of a water-mediated H-bonded network, which is required for proton-coupled electron transfer.

Chicken Egg Yolk as an Excellent Source of Highly Specific Antibodies Against Cytochromes P450

2004 ◽  
Vol 69 (3) ◽  
pp. 659-673 ◽  
Author(s):  
Petr Hodek ◽  
Tomáš Koblas ◽  
Helena Rýdlová ◽  
Božena Kubíčková ◽  
Miroslav Šulc ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cytochromes P450 ◽  
Egg Yolk ◽  
Good Alternative ◽  
Invasive Technique ◽  
Cytochrome P450 1A1 ◽  
Orconectes Limosus ◽  
Chicken Egg ◽  
Cytochrome P450 2B4 ◽  
Human Livers

Using chicken antibodies IgY (purified from egg yolks) against mammalian cytochromes P450 and by means of cytochrome P450 marker substrates, we found for the first time the presence of hepatopancreatic cytochrome P450 in crayfishOrconectes limosus(an inducible cytochrome P450 2B-like enzyme) and we were able to detect and quantify cytochrome P450 1A1 in microsomes of human livers. Expression levels of cytochrome P450 1A1 in human livers constituted less than 0.6% of the total hepatic cytochrome P450 complement. The results obtained in our study are clear examples that chicken IgY are suitable for cytochrome P450 detection and quantification. Due to the evolutionary distance, chicken IgY reacts with more epitopes on a mammalian antigen, which gives an amplification of the signal. Moreover, this approach offers many advantages over common mammalian antibody production since chicken egg is an abundant source of antibodies (about 100 mg IgY/yolk) and the egg collection is a non-invasive technique. In the case of antibodies against cytochrome P450 2B4, we documented fast and steady production of highly specific immunoglobulins. Thus, chicken antibodies should be considered as a good alternative to and/or superior substitute for conventional polyclonal antibody produced in mammals.

Five genetic polymorphisms of cytochrome P450 enzymes in the Czech non-Roma and Czech Roma population samples

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Lucie Dlouhá ◽  
Věra Adámková ◽  
Lenka Šedová ◽  
Věra Olišarová ◽  
Jaroslav A. Hubáček ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Genetic Polymorphisms ◽  
Metabolic Pathways ◽  
Cytochromes P450 ◽  
Taqman Assay ◽  
Cytochrome P450 Enzymes ◽  
Roma Population ◽  
Czech Population ◽  
Genotype Frequencies ◽  
Pcr Rflp

AbstractObjectivesCytochromes P450 play a role in human drugs metabolic pathways and their genes are among the most variable in humans. The aim of this study was to analyze genotype frequencies of five common polymorphisms of cytochromes P450 in Roma/Gypsy and Czech (non-Roma) population samples with Czech origin.MethodsRoma/Gypsy (n=302) and Czech subjects (n=298) were genotyped for CYP1A2 (rs762551), CYP2A6 (rs4105144), CYP2B6 (rs3745274) and CYP2D6 (rs3892097; rs1065852) polymorphisms using PCR-RFLP or Taqman assay.ResultsWe found significant allelic/genotype differences between ethnics in three genes. For rs3745274 polymorphism, there was increased frequency of T allele carriers in Roma in comparison with Czech population (53.1 vs. 43.7%; p=0.02). For rs4105144 (CYP2A6) there was higher frequency of T allele carriers in Roma in comparison with Czech population (68.7 vs. 49.8%; p<0.0001). For rs3892097 (CYP2D6) there was more carriers of the A allele between Roma in comparison with Czech population (39.2 vs. 38.2%; p=0.048). Genotype/allelic frequencies of CYP2D6 (rs1065852) and CYP1A2 (rs762551) variants did not significantly differ between the ethnics.ConclusionsThere were significant differences in allelic/genotype frequencies of some, but not all cytochromes P450 polymorphisms between the Czech Roma/Gypsies and Czech non-Roma subjects.

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