Detection of toxic effects of Cd2+ on different fish species via liver cytochrome P450-dependent monooxygenase activities and FTIR spectroscopy

The in vivo and in vitro effects of Cd²⁺ and the CYP1A inductor β-naphthoflavone(β-NF) on the hepatic cytochrome P450 (Cyt 450) monooxygenases were studied in silver carp (Hypophthalmichtys molitrix V.), wels (Silurus glanis L.), and carp (Cyprinus carpio). In vivo treatment of carp with a high dose of Cd²⁺ (10 mg kg⁻¹, for 3 days) caused a strong inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and a lower inhibition of 7-ethoxycoumarin-O-deethylase (ECOD) activity. The low-dose cadmium treatment (2 mg kg⁻¹ Cd²⁺, for 6+3 days) resulted in 4-fold increase in EROD and a 3-fold increase in ECOD activity. The combined treatment with Cd²⁺ and β-NF in both cases led to a loss of EROD inducibility. The silver carp and wels were treated with 10 mg L⁻¹ Cd²⁺ for 72 h in water. The Cyt P450 content in the wels liver microsomes was increased significantly after treatment for 48 h, whereas there was only a slight, not significant increase in Cyt P450 content in the silver carp microsomes. While the Cd²⁺ treatment resulted in inhibition of the CYP1A isoenzymes (EROD and ECOD), the APND (aminopyrene-N-demethylase, CYP2B or CYP3A isoenzyme) activity was increased 3- to 4-fold in both fish species. In vitro experiments of the effect of Cd²⁺ led to a concentration-dependent inhibition in all three investigated fish species. The ECOD isoenzyme of silver carp was the most sensitive to Cd²⁺. The lowest concentration of Cd²⁺ resulted in 50% inhibition. The APND isoenzyme was similarly sensitive to Cd²⁺ in all three investigated fish species. The most sensitive species was the wels, and the least sensitive were the carp isoenzyme. FTIR spectroscopy confirmed that cadmium caused damage to the protein structure. These results support the enzyme activity measurements measured in vivo and in vitro.     

细胞色素P450的电化学研究进展

细胞色素P450的电化学研究从一个侧面反映了为使细胞色素P450达到工业催化剂的最终目的人们所作的不懈努力。本文从细胞色素P450在电极上的电子转移研究,隧道扫描显微镜的微观成像研究和使用电极作为细胞色素P450的电子给体从而实现细胞色素P450底物转化三方面,评述了近年来细胞色素P450的电化学研究进展。     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at −2 and −278 mV (with a ratio of 1e⁻:3e⁻) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were −20 and −254 mV. All redox responses exhibit a pH dependence of approximately −59 mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry.     

Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X

Microbial cytochrome P450 enzymes (CYPs) are able to mimic the metabolism of human CYPs. One challenge is to identify the respective drug metabolites and to compare substrate specificities to those of the human enzymes. In this study, a class VIII self-sufficient CYP from Aspergillus fumigatus (CYP505X) and variants of this enzyme were heterologously expressed in E. coli. The substrate scope of the variants was determined using active pharmaceutical ingredients (APIs) and (hetero)cyclic compounds. Capsaicin – the active compound in chili peppers – was oxidized most efficiently (4.36?μM/min) in a whole cell mediated biotransformation. The products were isolated, purified and their structures elucidated by 1D and 2D NMR. The two major metabolites showed modifications on the lipophilic side chain. Specifically, capsaicin was hydroxylated at position 8 to give (E)-8-hydroxy-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide and epoxidized at the double bond to give N-(4-hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.     

Study of recombinant cytochrome P450 2C9 activity with diclofenac by MEKC

Cytochrome P450 2C9 (CYP2C9) is one of the most important isoforms in human liver involved in the metabolism of a large number of therapeutic agents. The aim of this paper is to demonstrate the applicability of CE for the determination of the enzymatic activity of CYP2C9 with diclofenac as a probe substrate. MEKC with SDS as a pseudostationary phase was used for this purpose. Compared to other assays, the MEKC-based method is rapid, can be automated and requires only a small quantity of enzymes and substrate. Moreover, the enzymatic reaction can be monitored with high sensitivity and repeatability even when the reaction mixture is used for the analysis without any pretreatment. The kinetic study on the given enzymatic reaction was also performed since the basic characterization of drug biotransformation generally begins with the enzyme kinetic analysis of metabolite formation. As a result, the Michaelis constant and maximum reaction velocity were evaluated, the values 3.44 +/- 0.45 microM and 19.78 +/- 0.76 nmol min(-1) nmol(-1), respectively, were in agreement with the literature data. On the other hand, a slight deviation from typical Michaelis-Menten kinetics with a weak positive cooperativity was found at diclofenac concentrations below 2 microM. The same atypical kinetic behavior of CYP2C9 was also observed by other authors.     

Effects of vindoline on rat cytochrome P450 isoform activities in vitro

A specific ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF–MS/MS) method has been described for the simultaneous determination of the metabolites of tacrine, bupropion, diclofenac, dextromethorphan and midazolam, which are the five probe drugs of the five cytochrome P450 (CYP450) isoforms CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A4. The inhibition degree was determined by calculating the IC₅₀. The chromatographic separation was performed on a C₁₈ column with a mobile phase consisting of 0.1% formic acid and acetonitrile. The mass spectrometric analysis was conducted in positive electrospray ionization mode. The IC₅₀ values of CYP1A2, CYP2B, CYP2C11, CYP2D1 and CYP3A were 113.4, 83.78, 22.50, 9.081 and 52.76 μmol L⁻¹, respectively. The in vitro results demonstrated that vindoline could inhibit CYP2D1 activity in rats, and weak inhibitory effect on CYP2C11 and CYP3A, but had no obvious effects on CYP1A2 and CYP2B.     

Structure-based design,synthesis of novel probes for cytochrome P450 OleT

Cytochrome P450 OleT_SA, a new cytochrome P450 enzyme from Staphylococcus aureus, catalyzes the oxidative decarboxylation and hydroxylation of fatty acids to generate terminal alkenes and fatty alcohols. The mechanism of this bifurcative chemistry remains largely unknown. Herein, a class of derivatized fatty acids were synthesized as probes to investigate the effects of substrate structure on the product type of P450 OleT_SA. The results demonstrate that the fine-tuned structure of substrates, even in a remote distance from the carboxyl group, significantly regulates OleT catalyzed decarboxylation/hydroxylation reactions. Molecular docking analysis indicated the potential interactions between the carboxylate groups of different probes and the enzyme active center which was attributed to the bifurcative chemistry.     

Electrochemical study of the interaction between cytochrome P450sccK201E and cholesterol

Cytochrome P450sccK201E, mutated form of cytochrome P450scc native recombinant (P450sccNR), was employed to study the enzyme–substrate interaction. The detection of the cholesterol was performed by electrochemical method using cyclic voltammetry (CV) and chronoamperometry measurements. The biochemical analysis was realized to observe the electrochemical responses of the engineerized enzyme to three different forms of cholesterol: free, low-density lipoprotein (LDL) and high-density lipoproteins (HDL). Compared to cytochrome P450sccNR, the cytochrome P450sccK201E displays a different behavior in the interaction with the substrate detection.

The results show that the engineerized enzyme can be utilized for the cholesterol detection in biosensor field.     

Molecular modeling of cytochrome P450 3A4

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.     

Non-covalent modification of the active site of cytochrome P450 for inverting the stereoselectivity of monooxygenation

The enantioselectivity in the sulfoxidation of thioanisole catalyzed by cytochrome P450_BSβ with a decoy molecule, a dummy molecule of the natural substrate, can be inverted by changing the structure of the decoy molecule. The methodology demonstrated herein shows the potential for controlling the stereoselectivity of biocatalysts without any mutagenesis.     

Rapid and accurate liquid chromatography and tandem mass spectrometry method for the simultaneous quantification of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes

The hepatic cytochrome P450 enzymes play a central role in the biotransformation of endogenous and exogenous substances. A sensitive high‐throughput liquid chromatography with tandem mass spectrometry assay was developed and validated for the simultaneous quantification of the products of ten metabolic reactions catalyzed by hepatic cytochrome P450 enzymes. After the substrates were incubated separately, the samples were pooled and analyzed by liquid chromatography with tandem mass spectrometry using an electrospray ionization source in the positive and negative ion modes. The method exhibited linearity over a broad concentration range, insensitivity to matrix effects, and high accuracy, precision, and stability. The novel method was successfully applied to study the kinetics of phenacetin‐O deethylation, coumarin‐7 hydroxylation, bupropion hydroxylation, taxol‐6 hydroxylation, omeprazole‐5 hydroxylation, dextromethorphan‐O demethylation, tolbutamide‐4 hydroxylation, chlorzoxazone‐6 hydroxylation, testosterone‐6β hydroxylation, and midazolam‐1 hydroxylation in rat liver microsomes.     

A comparative molecular field analysis of cytochrome P450 2A5 and 2A6 inhibitors

Structure-activity relationships of 23 P450 2A5 and 2A6 inhibitors were analysed using the CoMFA [1] and GOLPE/GRID with smart region definition (SRD) [2]. The predictive power of the resulting models was validated using five compounds not belonging to the model set. All models have high internal and external predictive power and resulting 3D-QSAR models are supporting each other. Both Sybyl and GOLPE highlight properties near lactone moiety to be important for 2A5 and 2A6 inhibition. Another important feature for pIC₅₀ was the size of the substituent in the 7-positon of coumarin. The models suggest that the 2A5 binding site is larger that that of 2A6 due to larger steric regions in the CoMFA coefficient maps and corresponding GOLPE maps. In addition, the maps reveal that 2A6 disfavours negative charge near the lactone moiety of coumarin.     

Hartree–Fock and density functional theory calculations for the reaction mechanism of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum

A reaction mechanism of a nitric oxide reductase, cytochrome P450nor (P450nor) from Fusarium oxysporum, was clarified by using Density functional theory and Hartree–Fock calculations. In this reaction mechanism, molecular orbital (MO) analysis revealed that the NO ligand dissociates from the heme iron immediately after one-electron reduction by NADH, and MO energy analysis revealed that NADH acts as a one-electron reducer, not as a two-electron reducer, and that NADH has a pivotal role different from other one-electron reducers. The role of NADH is to act as a double one-electron donor (i.e. one-electron transfer occurring twice) and to combine with the NO⁻ molecule by charge recombination reaction. Our quantum chemical calculations indicated that all reactions occurring in the heme pocket are too fast to become rate-limiting. Therefore, the rate-limiting steps in the proposed reaction mechanism are the process of capturing NO and NADH into the heme pocket and the process of expelling a product generated in the heme pocket. Kinetics of these processes was discussed based on large-amplitude vibration, which helps capturing and expelling processes in a widely opened heme pocket of P450nor. The reaction mechanism proposed here well explains published experimental data.     

Spin state transitions in Langmuir–Blodgett films of recombinant cytochrome P450scc and adrenodoxin

Langmuir–Blodgett (LB) films of recombinant cytochrome P450scc, of P450scc–adrenodoxin (Adx) complex and alternated layers of Adx and P450scc have been obtained. Spectral properties of these proteins in thin films were investigated by UV–Vis absorption spectroscopy. It has been found that cytochrome P450scc exists in LB films only in low-spin state while before the deposition it was in high-spin state. The data suggest that transferring the hemoprotein or its complex with redox partner results in the modification of the spin state by a conformational transition. In order to investigate further the P450scc and Adx interaction, the mass density of the films formed from these molecules has been studied by nanogravimetric measurements. Comparative study between nanogravimetric and spectral characterisation was performed. The results indicate that the protein–protein interaction is disrupted, when the complex is organised in thin film.     

The stereochemistry of fatty acid hydroxylation by cytochrome P450BM3

The stereochemical preference for the cytochrome P450_BM3-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 P450_BM3 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 P450_BM3.     

Direct electrochemistry of cytochrome P450 27B1 in surfactant films

We report the first direct electrochemistry of cytochrome P450 27B1 (CYP27B1) immobilized on an edge-plane pyrolytic graphite (EPG) electrode coated with a film of didodecyldimethylammonium bromide (DDAB). Cyclic voltammetry (CV) in a deoxygenated solution revealed excellent electrochemical reversibility with an average midpoint potential of −180 ± 5 mV vs. Ag/AgCl and an apparent surface coverage of (7.0 ± 2.5) × 10¹³ molecules per cm². The rate of heterogeneous electron transfer between CYP27B1 and the EPG electrode was determined to be 3.5 ± 0.6 s⁻¹. Upon addition of oxygen, a significant increase in cathodic current occurred, likely due to electrocatalytic reduction of dioxygen to peroxide and/or water by CYP27B1. Characterization of the electrochemical properties of CYP27B1 is an important first step toward developing a bioelectrochemical method for measuring vitamin D in serum.     

Study of atypical kinetic behaviour of cytochrome P450 2C9 isoform with diclofenac at low substrate concentrations by sweeping‐MEKC combination

In view of the fact that several studies have shown that diclofenac hydroxylation by cytochrome P450 2C9 deviated from Michaelis–Menten kinetics at low substrate concentrations, sweeping combined with MEKC was applied for the kinetic study of this pharmacologically important reaction. A 50 μm fused silica capillary (56 cm effective length) was used to carry out all separations. 70 mM SDS in 20 mM phosphate 20 mM tetraborate buffer, pH 8.6, was used as the BGE. Injection was accomplished by the application of 50 mbar (5 kPa) pressure to the sample vial for 52 s. Separation was performed at 22 kV (positive polarity), with a capillary temperature of 25°C and detection at 200 nm. The higher sensitivity of the sweeping‐MEKC combination compared with the simple MEKC method enabled this reaction to be fitted to a Hill kinetic model and confirmed the findings of other authors. A Michaelis constant of 2.91±0.10 μM, maximum reaction velocity of 9.16±0.16 nmol/min/nmol and Hill coefficient of 1.66±0.08 were determined. This value of Hill coefficient confirms the presence of a positive cooperativity at low diclofenac concentrations and supports the hypothesis of two substrates binding at or near the active site.     

Intracellular transport and localization of microsomal cytochrome P450

The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH₂-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.     

A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions.