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.

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 Complex structure of NADPH-cytochrome P450 reductase and heme oxygenase-1

2014 ◽  
Vol 70 (a1) ◽  
pp. C478-C478
Author(s):  
Masakazu Sugishima ◽  
Hideaki Sato ◽  
Yuichiro Higashimoto ◽  
Jiro Harada ◽  
Kei Wada ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Heme Oxygenase ◽  
Complex Structure ◽  
Stable Complex ◽  
Heme Oxygenase 1 ◽  
Closed Conformation ◽  
Nadph Cytochrome ◽  
Redox Partner ◽  
Open Conformation

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to FAD in CPR, then to FMN in CPR, and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, ``closed-open transition'' of CPR is indispensable because FMN in the closed conformation of CPR is covered by FAD-binding domain, thus FMN is not exposed to the surface in the closed conformation. Recently, Hamdane et al. determined the crystal structures of a hinge-shortened rat CPR variant (ΔTGEE), which favors an open conformation [1]. In the open conformation of CPR, FMN is exposed to the surface, thus this conformation appears to be favorable to interact with the redox partners, though no complex structure of CPR and its redox partner has been determined. Here, we demonstrate that ΔTGEE makes a stable complex with heme-rat HO-1 (rHO-1) complex and can support HO reaction, though its efficiency is extremely limited. Further we determine the crystal structure of ΔTGEE in complex with heme-rHO-1 at 4.3 Å resolution [2]. X-ray scattering and biochemical data suggest that the complex structure of ΔTGEE and heme-rHO-1 is similar to that of wild type CPR and heme-rHO-1. Distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme. On the other hand, FAD is far from FMN and heme, indicating that the ``closed-open transition'' of CPR is required for electron transfer from FAD to FMN.

scholarly journals Nitric oxide is a mediator of the decrease in cytochrome P450-dependent metabolism caused by immunostimulants

1993 ◽  
Vol 90 (23) ◽  
pp. 11147-11151 ◽  
Author(s):  
O G Khatsenko ◽  
S S Gross ◽  
A B Rifkind ◽  
J R Vane
Keyword(s):  
Nitric Oxide ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Cytochromes P450 ◽  
Hepatic Metabolism ◽  
Spectral Studies ◽  
Rat Liver Microsomes ◽  
Hepatic Microsomes ◽  
Oxide Synthase

Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixed-function oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now known to cause NO overproduction by cells via induction of nitric oxide synthase. We have investigated whether NO overproduction is involved in suppressing hepatic metabolism by LPS. In vitro treatment of hepatic microsomes with NO, produced by chemical decomposition of 3-morpholinosydnonimine or by nitric oxide synthase, substantially suppressed cytochrome P450-dependent oxygenation reactions. This effect of NO was seen with hepatic microsomes prepared from two species (rat and chicken) and after exposure to chemicals that induce distinct molecular isoforms of cytochromes P450 (beta-naphthoflavone, 3-methylcholanthrene, and phenobarbital). Spectral studies indicate that NO reacts in vitro with both Fe(2+)- and Fe(3+)-hemes in microsomal cytochromes P450. In vivo, LPS diminished the phenobarbital-induced dealkylation of 7-pentoxyresorufin by rat liver microsomes and reduced the apparent P450 content as measured by CO binding. These LPS effects were associated with induction of NO synthesis; LPS-induced NO synthesis showed a strong positive correlation with the severity of cytochrome P450 inhibition. The decrease in both hepatic microsomal P450 activity and CO binding caused by LPS was largely prevented by the selective NO synthase inhibitor N omega-nitro-L-arginine methyl ester. Our findings implicate NO over-production as a major factor mediating the suppression of hepatic metabolism by immunostimulants such as LPS.

scholarly journals Synthesis of triazole-containing ciprofloxacin conjugate and its in silico test as a cytochrome P450 ligand

2021 ◽  
pp. 21-27
Author(s):  
Ярослав Вячеславович Фалетров ◽  
Лилия Игоревна Глинская ◽  
Матвей Сергеевич Хорецкий ◽  
Ян Владимирович Панада ◽  
Нина Степановна Фролова ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Virtual Screening ◽  
High Throughput ◽  
Active Site ◽  
Essential Role ◽  
Cytochromes P450 ◽  
Heme Iron ◽  
Cellular Functions ◽  
Docking Calculations

Cytochromes P450 are hem-containing monooxygenases which catalyse biosynthesis of many compounds playing an essential role in cellular functions as well as degradation of drugs and xenobiotics. Some P450s (e. g., human CYP19 and CYP17, fungal CYP51) are valid target proteins for some drugs. The others P450s are also interesting for pharmacology-related researches. Aiming to design new fluorescent inhibitor of P450s we have synthesised the azole-bearing conjugate of ciprofloxacin (CPF-bab-Z1). To estimate potential of the compound as a ligand for CYPs we performed high-throughput virtual screening (multiple docking calculations) for CPF-bab-Z1 and multiple known 3D structures of P450s. The best affinity for CPF-bab-Z1 (the smallest value of energy of binding is equal -12.5 kcal/mol) were found for protein with PDB code 5esh among 28 structures of CYP51. The calculated pose of CPF-bab-Z1 in the active site of the protein is characterised by cyclopropyl (but not azole) proximity to the heme iron of the CYP51. The data obtained demonstrate perspectives for in vitro investigations of CPF-bab-Z1 with P450s.

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.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

2020 ◽  
Vol 27 (3) ◽  
pp. 201-209
Author(s):  
Syed Saqib Ali ◽  
Mohammad Khalid Zia ◽  
Tooba Siddiqui ◽  
Haseeb Ahsan ◽  
Fahim Halim Khan
Keyword(s):  
Ascorbic Acid ◽  
Conformational Changes ◽  
Structural Changes ◽  
The Body ◽  
Titration Calorimetry ◽  
Spectroscopic Techniques ◽  
Human Beings ◽  
Plasma Ascorbic Acid ◽  
Dietary Antioxidant ◽  
And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

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.

scholarly journals In Silico Prediction of Drug–Drug Interactions Mediated by Cytochrome P450 Isoforms

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 538
Author(s):  
Alexander V. Dmitriev ◽  
Anastassia V. Rudik ◽  
Dmitry A. Karasev ◽  
Pavel V. Pogodin ◽  
Alexey A. Lagunin ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Drug Interactions ◽  
Cytochromes P450 ◽  
Investigational Drug ◽  
Web Resource ◽  
Average Accuracy ◽  
Structural Formulas ◽  
Cytochrome P450 Isoforms ◽  
Leave One Out ◽  
Important Drug

Drug–drug interactions (DDIs) can cause drug toxicities, reduced pharmacological effects, and adverse drug reactions. Studies aiming to determine the possible DDIs for an investigational drug are part of the drug discovery and development process and include an assessment of the DDIs potential mediated by inhibition or induction of the most important drug-metabolizing cytochrome P450 isoforms. Our study was dedicated to creating a computer model for prediction of the DDIs mediated by the seven most important P450 cytochromes: CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, and CYP3A4. For the creation of structure–activity relationship (SAR) models that predict metabolism-mediated DDIs for pairs of molecules, we applied the Prediction of Activity Spectra for Substances (PASS) software and Pairs of Substances Multilevel Neighborhoods of Atoms (PoSMNA) descriptors calculated based on structural formulas. About 2500 records on DDIs mediated by these cytochromes were used as a training set. Prediction can be carried out both for known drugs and for new, not-yet-synthesized substances. The average accuracy of the prediction of DDIs mediated by various isoforms of cytochrome P450 estimated by leave-one-out cross-validation (LOO CV) procedures was about 0.92. The SAR models created are publicly available as a web resource and provide predictions of DDIs mediated by the most important cytochromes P450.

Sign in / Sign up

Export Citation Format

Share Document