共查询到20条相似文献，搜索用时 15 毫秒
1.
Yuri D. Ivanov Amir Taldaev Andrey V. Lisitsa Elena A. Ponomarenko Alexander I. Archakov 《Molecules (Basel, Switzerland)》2022,27(4)
The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (ket), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor β, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about kcat. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly, kcat. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation. 相似文献
2.
《Electroanalysis》2017,29(7):1674-1682
Human cytochrome CYP1A2 is one of the major hepatic cytochrome P450s involved in many drugs metabolism, and chemical carcinogens activation. The CYP1A2‐dsDNA interaction in situ evaluation using a DNA‐electrochemical biosensor and differential pulse voltammetry was investigated. A dsDNA‐electrochemical biosensor showed that CYP1A2 interacted with dsDNA causing conformational changes in the double helix chain and DNA oxidative damage. A preferential interaction between the dsDNA guanosine residues and CYP1A2 was found, as free guanine and 8‐oxoguanine, a DNA oxidative damage biomarker, oxidation peaks were detected. This was confirmed using guanine and adenine homopolynucleotides‐electrochemical biosensors. The CYP1A2‐dsDNA interaction and dsDNA conformation changes was also confirmed by UV‐Vis spectrophotometry. 相似文献
3.
Schumann CA Dörrenhaus A Franzke J Lampen P Dittrich PS Manz A Roos PH 《Analytical and bioanalytical chemistry》2008,392(6):1159-1166
To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most
appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ,
however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties
of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the
human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells,
we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It
is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin
O-deethylase (EROD). This is also true for the cells’ CYP1A1 protein content. With some exceptions, the EROD activity largely
coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising
and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the
biochemical and molecular heterogeneity of cells.
Figure Formation of fluorescent resorufin from ethoxyresorufin by cytochrome P450 activity in urothelial cells attached within the
chamber of a microfluidic device 相似文献
4.
Max J. Cryle 《Tetrahedron letters》2007,48(1):133-136
The stereochemical preference for the cytochrome P450BM3-catalysed hydroxylation of tetradecanoic and pentadecanoic acids has been determined via comparison with authentic non-racemic standards utilising enantioselective HPLC. The sub-terminal hydroxylation of these fatty acids by P450BM3 is highly selective for the formation of the R-alcohols. This is the same enantioselectivity as is seen for hexadecanoic acid oxidation but contrasts with a previous report of S-hydroxylation of pentadecanoic acid by P450BM3. 相似文献
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7.
Jung C 《Analytical and bioanalytical chemistry》2008,392(6):1031-1058
Cytochrome P450 proteins (CYPs) are a big class of heme proteins which are involved in various metabolic processes of living
organisms. CYPs are the terminal catalytically active components of monooxygenase systems where the substrate binds and is
hydroxylated. In order to be functionally competent, the protein structures of CYPs possess specific properties that must
be explored in order to understand structure–function relationships and mechanistic aspects. Fourier transform infrared spectroscopy
(FTIR) is one tool that is used to study these structural properties. The application of FTIR spectroscopy to the secondary
structures of CYP proteins, protein unfolding, protein–protein interactions and the structure and dynamics of the CYP heme
pocket is reviewed. A comparison with other thiolate heme proteins (nitric oxide synthase and chloroperoxidase) is also included.
Figure The protein secondary structure, protein unfolding, redox-partner protein–protein interaction, structural changes induced
by the reduction of the heme iron, and the structure and dynamics of the active site of cytochromes P450 (CYP) can be studied
using Fourier transform infrared spectroscopy (FTIR). FTIR spectroscopy is a good approach for gaining a deeper insight into
structure–function relationships in CYPs.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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9.
Abayomi S. Faponle Anupom Roy Ayodeji A. Adelegan James W. Gauld 《Molecules (Basel, Switzerland)》2021,26(12)
Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl’s side chain tightens and relaxes the opening to the channel in conjunction with the arginyl’s, though the latter’s side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel. 相似文献
10.
表面增强拉曼光谱:应用和发展 总被引：2，自引：0，他引：2
表面增强拉曼光谱技术(Surface-enhanced Raman spectroscopy,SERS)是一种具有超高灵敏度的指纹光谱技术,目前已广泛应用于表面科学、材料科学、生物医学、药物分析、食品安全、环境检测等领域,是一种极具潜力的痕量分析技术。 本文对SERS技术及相关的针尖增强拉曼光谱(Tip-enhanced Raman spectroscopy,TERS),壳层隔绝纳米粒子增强拉曼光谱(Shell-isolated nanoparticle-enhanced Raman spectroscopy,SHINERS)技术的发展及应用进行了综合评述,并探讨了其未来的研究热点及发展方向。 相似文献
11.
Dr. Célia M. Silveira Dr. Patrícia R. Rodrigues Dr. Wissam Ghach Dr. Sofia A. Pereira Dr. Francisco Esteves Dr. Michel Kranendonk Dr. Mathieu Etienne Prof. Dr. M. Gabriela Almeida 《ChemElectroChem》2021,8(3):500-507
The direct electrochemical response of membrane-bound human cytochrome P450 1A2 (CYP1A2) was studied on pyrolytic graphite electrodes, while encapsulated in a sol-gel matrix. The enzymatic reduction of O2 was evaluated in the presence and absence of its electron donor partner, cytochrome P450 oxidoreductase (CPR). When used without CPR, CYP1A2 was shown to be highly sensitive to O2 even in the presence of residual amounts. Under aerobic conditions (air-saturated solutions), the catalytic signal attributed to the reaction with O2 was lost, suggesting the enzyme was inactivated. In contrast, the CYP1A2/CPR complex retained O2 reductase activity with high O2 concentration in solution. The results demonstrated a crucial role of CPR in stabilizing the immobilized CYP1A2 enzyme and in the preservation of O2 electrocatalysis, when using this electrochemical set-up. Though the enzyme's monooxygenase activity towards caffeine was not detected, this study highlights the complexity of coupling CYP1A2 reduction currents with substrate turnover, owing to the simultaneous electrochemical measurement of the O2 reduction reaction. 相似文献
12.
《Analytical letters》2012,45(17):2549-2561
Caffeine is a useful indicator to quickly assess liver function. High-throughput tests are needed for single-point caffeine measurements, with low cross-reactivity toward its major metabolite, paraxanthine. A newly developed ELISA was compared with an LC-MS/MS reference method, using 60 saliva samples from 10 individuals, before and after caffeine intake. Bland-Altman plot, Student t-test and F-test were used to compare the two methods. Proteins were precipitated using organic solvent and the caffeine recoveries compared with those obtained using sample microfiltration. The antibody, with a low cross-reactivity toward paraxanthine (0.08%), allows quantification of caffeine in saliva samples from 2.5 µg/L to 125 µg/L with high precision and the ELISA shows comparable results to those obtained by LC-MS/MS. A one-step protein precipitation using an organic solvent provides comparable results to a more costly and time-consuming microfiltration pre-treatment of samples. The new ELISA is a fit-for-purpose method to accurately and precisely determine caffeine in saliva samples. 相似文献
13.
The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body’s immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug’s metabolism in vivo. 相似文献
14.
Ignacio Viciano Raquel Castillo Sergio Martí 《Journal of computational chemistry》2015,36(23):1736-1747
CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three‐step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone‐dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydroxylated products through a highly exergonic reaction. In addition, an analysis of the primary deuterium kinetic isotopic effects, calculated for the hydrogen abstraction step, reveals values (～10) overpassing the semiclassical limit for the C? H, indicating the presence of a substantial tunnel effect. Finally, a decomposition analysis of the interaction energy for the substrate and cofactor in the active site is also discussed. According to our results, the role of the enzymatic environment consists of a transition state stabilization by means of dispersive and polarization effects. © 2015 Wiley Periodicals, Inc. 相似文献
15.
Li MD Yeung CS Guan X Ma J Li W Ma C Phillips DL 《Chemistry (Weinheim an der Bergstrasse, Germany)》2011,17(39):10935-10950
We present an investigation of the decarboxylation reaction of ketoprofen (KP) induced by triplet excited-state intramolecular proton transfer in water-rich and acidic solutions. Nanosecond time-resolved resonance Raman spectroscopy results show that the decarboxylation reaction is facile in aqueous solutions with high water ratios (water/acetonitrile ≥50%) or acidic solutions with moderate and strong acid concentration. These experimental results are consistent with results from density functional theory calculations in which 1) the activation energy barriers for the triplet-state intramolecular proton transfer and associated decarboxylation process become lower when more water molecules (from one up to four molecules) are involved in the reaction system and 2) perchloric acid, sulfuric acid, and hydrochloric acid can shuttle a proton from the carboxyl to carbonyl group through an initial intramolecular proton transfer of the triplet excited state, which facilitates the cleavage of the C-C bond, thus leading to the decarboxylation reaction of triplet state KP. During the decarboxylation process, the water molecules and acid molecules may act as bridges to mediate intramolecular proton transfer for the triplet state KP when KP is irradiated by ultraviolet light in water-rich or acidic aqueous solutions and subsequently it generates a triplet-protonated carbanion biradical species. The faster generation of triplet-protonated carbanion biradical in acidic solutions than in water-rich solutions with a high water ratio is also supported by the lower activation energy barrier calculated for the acid-mediated reactions versus those of water-molecule-assisted reactions. 相似文献
16.
曾敏静;马玮玮;唐浴尘;高婷娟 《分析测试学报》2024,43(1):95-106
细胞成像在生命科学与药物研究中具有重要意义。拉曼光谱作为一种非破坏性的振动光谱技术,结合非标记或探针标记技术可对细胞不同组分进行成像。由于自发拉曼光谱信号较弱,运用有效的增强手段可提高细胞成像的时间与空间分辨率。该文综述了表面增强拉曼光谱（SERS）、相干拉曼光谱（CRS）、共振拉曼光谱（RRS）等拉曼增强方法在线粒体、溶酶体和内质网等细胞器成像中的研究进展,以及上述方法在蛋白质、脂质、糖类和核酸等重要细胞生物分子成像中的应用。此外,还讨论了标记技术中拉曼探针的化学结构、增强因子、检出限等因素对细胞成像的影响,并分析了当前细胞拉曼成像的发展趋势、存在的挑战和可能的解决方案。 相似文献
17.
Supranee Wiwatchaiwong Hirotoshi Matsumura Nobuhumi Nakamura Masafumi Yohda Hiroyuki Ohno 《Electroanalysis》2007,19(5):561-565
We have studied the characterization of thermophilic cytochrome P450 (P450st)‐didodecyldimethylammonium bromide (DDAB) films by using UV‐vis absorption, resonance Raman spectroscopy, and electrochemical methods. The observed Raman spectrum indicated near‐native conformation of the heme iron in DDAB film on the surface of a glass slide, while on the surface of a plastic‐formed carbon (PFC) electrode, the conformation of P450st‐DDAB was very similar to that of heme‐DDAB film, suggesting the release of heme from P450st in DDAB films on PFC electrodes. When NaBr was added as salt to the casting solution, the result of Raman spectrum indicated near‐native conformation of P450st in DDAB film even on the PFC electrode, but no redox potential of P450st which has near native structure was observed. This study suggests the essential experimental conditions when working with heme protein‐DDAB films as, in some cases, heme iron from proteins is released on the surface of the electrode. 相似文献
18.
Richard A. Mathies 《Journal of Chemical Sciences》1991,103(3):283-293
Bacteriorhodopsin is a retinal-containing protein that functions as a light-driven proton pump. Resonance Raman and femtosecond
dynamic absorption spectroscopy are being used to elucidate the molecular mechanism of bacteriorhodopsin. The primary photochemical
process is atrans- to-cis isomerization about the C13=C14 bond of the retinal chromophore that has been directly observed using femtosecond dynamic absorption spectroscopy. The excited
state isomerization dynamics can be quantitatively analyzed using a new theory for nonstationary state spectroscopy. Resonance
Raman vibrational spectroscopy has been used to determine the structure of the chromophore in each of bacteriorhodopsin’s
intermediates and to analyze the kinetics of the photocycle. These results are integrated into an explicit molecular model
(the C-T Model) for proton pumping in bacteriorhodopsin. 相似文献
19.
Dr. Jungkweon Choi Sachiko Tojo Dr. Doo-Sik Ahn Prof. Dr. Mamoru Fujitsuka Shunichi Miyamoto Prof. Dr. Kazuo Kobayashi Prof. Dr. Hyotcherl Ihee Prof. Dr. Tetsuro Majima 《Chemistry (Weinheim an der Bergstrasse, Germany)》2019,25(32):7711-7718
Despite numerous experimental and theoretical studies, the proton transfer accompanying the oxidation of 2′-deoxyadenosine 5′-monophosphate 2’-deoxyadenosine 5’-monophosphate (5’-dAMP, A ) is still under debate. To address this issue, we have investigated the oxidation of A in acidic and neutral solutions by using transient absorption (TA) and time-resolved resonance Raman (TR3) spectroscopic methods in combination with pulse radiolysis. The steady-state Raman signal of A was significantly affected by the solution pH, but not by the concentration of adenosine (2–50 mm ). More specifically, the A in acidic and neutral solutions exists in its protonated ( A H+(N1+H+)) and neutral ( A ) forms, respectively. On the one hand, the TA spectral changes observed at neutral pH revealed that the radical cation ( A .+) generated by pulse radiolysis is rapidly converted into A .(N6−H) through the loss of an imino proton from N6. In contrast, at acidic pH (<4), A H.2+(N1+H+) generated by pulse radiolysis of A H+(N1+H+) does not undergo the deprotonation process owing to the pKa value of A H.2+(N1+H+), which is higher than the solution pH. Furthermore, the results presented in this study have demonstrated that A , A H+(N1+H+), and their radical species exist as monomers in the concentration range of 2–50 mm . Compared with the Raman bands of A H+(N1+H+), the TR3 bands of A H.2+(N1+H+) are significantly down-shifted, indicating a decrease in the bond order of the pyrimidine and imidazole rings due to the resonance structure of A H.2+(N1+H+). Meanwhile, A .(N6−H) does not show a Raman band corresponding to the pyrimidine+NH2 scissoring vibration due to diprotonation at the N6 position. These results support the final products generated by the oxidation of adenosine in acidic and neutral solutions being A H.2+(N1+H+) and A .(N6−H), respectively. 相似文献
20.
Dr. Long Qu Pei Liu Xiaolu Tian Chengyong Shu Yikun Yi Pu Yang Te Wang Binren Fang Prof. Mingtao Li Prof. Bolun Yang 《ChemElectroChem》2020,7(7):1679-1688
A pomegranate-like cathode, VN/S@G, is synthesized according to a simple principle of electrostatic attraction through a controllable Zeta potential method, establishing a hierarchical-structured VN/S nanoclusters encapsuled with graphene nanosheets. Internal VN nanoparticles trap lithium polysulphides (LiPSs) and catalyse them transforming from long-chain to short-chain species; whereas the external cladding layers of graphene nanosheets confine the transformations in a nanoscale-catalysis reactors. VN/S@G cathode exhibits excellent long-cycling life at 2 C rate during the 2000 cycles, corresponding to 0.038 percent of capacity fade per cycle. According to in-situ Raman and electrochemical impedance spectroscopies, VN catalyst accelerates chemical transformations of liquid-state LiPSs to solid-state Li2S2/Li2S and graphene intensifies Li+ diffusion behaviour. Improvement of electrochemical performance of the VN/S@G cathode depends on a coefficient of physical and chemical interactions between VN catalyst and LiPSs species. 相似文献