Mammalian cytochromes P450--importance of tissue specificity

Mammals express multiple cytochromes P450 simultaneously in a variety of tissues, including the liver, kidney, lung, adrenal, gonads, brain, and most others. For cytochromes P450 that are expressed in many tissues or cell types, the tissue/cell type-specific expression might be associated with their special physiological roles. Several cytochrome P450 enzymes are found not only in different cell types and tissues, but also in different subcellular compartments. Generally, all mammalian cytochrome P450 enzymes are membrane bound. The two major groups are represented by microsomal cytochromes P450 that reside in the endoplasmic reticulum, and mitochondrial cytochromes P450, that reside in the inner mitochondrial membrane. However, the outer nuclear membrane, different Golgi compartments, peroxisomes and the plasma membrane are also sites where cytochromes P450 were observed. For example, CYP51 is an ER enzyme in majority of tissues but in male germ cells it trafficks through the Golgi to acrosome, where it is stabilized for several weeks. Surprisingly, in brains of heme synthesis deficient mice, a soluble form of CYP1A1 was detected whose activity has been restored by the addition of heme. In the majority of cases each cytochrome P450 enzyme resides in a single subcellular compartment in a certain cell, however, examples of simultaneous localization in different subcellular compartments have also been described, such as endoplasmic reticulum, Golgi and plasma membrane for CYP2E1. This review will focus on the physiological importance of mammalian cytochrome P450 expression and localization in different tissues or cell types and subcellular compartments.     

Metabolons involving plant cytochrome P450s

Arranging biological processes into “compartments” is a key feature of all eukaryotic cells. Through this mechanism, cells can drastically increase metabolic efficiency and manage complex cellular processes more efficiently, saving space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cytochrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabolons around P450s on the surface of the ER. We␣outline direct and indirect experimental data that describes P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors.     

New findings in studies of cytochromes P450

Cytochromes P450 represent a numerous family of heme-containing enzymes belonging to the group of monooxygenases. In prokaryotes, cytochromes P450 usually perform a plastic function, whereas in eukaryotes their functions are very diverse. Mammalian cytochromes P450 are components of membranes and are involved in biosynthesis and metabolism of many physiologically active substances; moreover, these cytochromes are unique in their ability to catalyze biotransformation of xenobiotics, i.e. metabolize substances of foreign origin (drugs, toxins, environmental pollutants). The latter promotes elimination of xenobiotics, but sometimes intermediates of their metabolism are even more toxic and dangerous than the original xenobiotics per se. Some catalytic features of cytochromes P450 still need unambiguous explanation, i.e. broad substrate specificity, diversity of catalytic reactions, and unusual kinetics. Under some conditions, cytochromes P450 can produce reactive oxygen species, and this is another problem attracting increasing attention. In this respect, a recent finding in mitochondria of analogs of microsomal cytochromes P450 seems especially intriguing; it was postulated that P450 can be responsible for mitochondrial dysfunction, cell apoptosis, and pathogenesis of some diseases. In this paper the present state of the art concerning these problems is considered.     

Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450.

Flavonoids represent a group of phytochemicals exhibiting a wide range of biological activities arising mainly from their antioxidant properties and ability to modulate several enzymes or cell receptors. Flavonoids have been recognized to exert anti-bacterial and anti-viral activity, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, hepatoprotective, cytostatic, apoptotic, estrogenic and anti-estrogenic properties. However, not all flavonoids and their actions are necessarily beneficial. Some flavonoids have mutagenic and/or prooxidant effects and can also interfere with essential biochemical pathways. Among the proteins that interact with flavonoids, cytochromes P450 (CYPs), monooxygenases metabolizing xenobiotics (e.g. drugs, carcinogens) and endogenous substrates (e.g. steroids), play a prominent role. Flavonoid compounds influence these enzymes in several ways: flavonoids induce the expression of several CYPs and modulate (inhibit or stimulate) their metabolic activity. In addition, some CYPs participate in metabolism of flavonoids. Flavonoids enhance activation of carcinogens and/or influence the metabolism of drugs via induction of specific CYPs. On the other hand, inhibition of CYPs involved in carcinogen activation and scavenging reactive species formed from carcinogens by CYP-mediated reactions can be beneficial properties of various flavonoids. Flavonoids show an estrogenic or anti-estrogenic activity owing to the structural similarity with the estrogen skeleton. Mimicking natural estrogens, they bind to estrogen receptor and modulate its activity. They also block CYP19, a crucial enzyme involved in estrogen biosynthesis. Flavonoids in human diet may reduce the risk of various cancers, especially hormone-dependent breast and prostate cancers, as well preventing menopausal symptoms. For these reasons the structure-function relationship of flavonoids is extensively studied to provide an inspiration for a rational drug and/or chemopreventive agent design of future pharmaceuticals.     

Prediction of binding modes for ligands in the cytochromes P450 and other heme-containing proteins

The cytochromes P450 (P450s) are a family of heme-containing monooxygenase enzymes involved in a variety of functions, including the metabolism of endogenous and exogenous substances in the human body. During lead optimization, and in drug development, many potential drug candidates are rejected because of the affinity they display for drug-metabolising P450s. Recently, crystal structures of human enzymes involved in drug metabolism have been determined, significantly augmenting the prospect of using structure-based design to modulate the binding and metabolizing properties of compounds against P450 proteins. An important step in the application of structure-based metabolic optimization is the accurate prediction of docking modes in heme binding proteins. In this paper we assess the performance of the docking program GOLD at predicting the binding mode of 45 heme-containing complexes. We achieved success rates of 64% and 57% for Chemscore and Goldscore respectively; these success rates are significantly lower than the value of 79% observed with both scoring functions for the full GOLD validation set. Re-parameterization of metal-acceptor interactions and lipophilicity of planar nitrogen atoms in the scoring functions resulted in a significant increase in the percentage of successful dockings against the heme binding proteins (Chemscore 73%, Goldscore 65%). The modified scoring functions will be useful in docking applications on P450 enzymes and other heme binding proteins.     

Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts

Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.     

赤点石斑鱼两种芳香化酶cDNA的克隆及其表达的组织特异性

以赤点石斑鱼 (Epinephelusakaara)脑垂体中提取的RNA为模板 ,根据芳香化酶的保守序列设计引物 ,利用GeneRacerTM 技术 ,克隆出两种芳香化酶即脑芳香化酶 (P4 5 0aromB)和性腺芳香化酶 (P4 5 0aromA)的cDNA ,其全长分别为 190 1bp (编码 5 0 9aa)和 1833bp (编码 5 18aa)。序列分析结果表明 ,赤点石斑鱼两种芳香化酶cDNA序列的同源性为 5 1 6 % ,氨基酸序列之间同源性为 6 2 5 % ,与斜带石斑鱼两种芳香化酶氨基酸同源性分别为 94 7%和 97 9%。对 8个科的 10种鱼进行了分子系统进化树分析 ,结果与根据传统的形态学和生化特征分类进化地位基本一致。以特异性引物扩增雌、雄赤点石斑鱼各种组织 (垂体、嗅球、端脑、下丘脑、中脑、后脑、延脑、心脏、肾脏、肝脏、脾脏、性腺、鳃、胃、肠、皮肤、脂肪、肌肉、头肾、胸腺、鳔 ) ,以β actin作内标比较各组织芳香化酶基因表达量的差异 ,结果表明 ,赤点石斑鱼脑芳香化酶 (P4 5 0aromB)有广泛的组织分布 ,脑和垂体的表达量很高 ,各组织表达量有明显的雌、雄差异 ;而性腺芳香化酶 (P4 5 0aromA)表达主要集中于垂体和性腺 ,且不论雌雄 ,其性腺表达量均高于脑垂体 ,和P4 5 0aromB的表达模式明显不同 ,表现为在脑部 ,P4 5 0aromB表达量高于P4 5 0aromA ,而在性腺 ,     

Cyclopropylamine inactivation of cytochromes P450: role of metabolic intermediate complexes

The inactivation of cytochrome P450 enzymes by cyclopropylamines has been attributed to a mechanism involving initial one-electron oxidation at nitrogen followed by scission of the cyclopropane ring leading to covalent modification of the enzyme. Herein, we report that in liver microsomes N-cyclopropylbenzylamine (1) and related compounds inactivate P450 to a large extent via formation of metabolic intermediate complexes (MICs) in which a nitroso metabolite coordinates tightly to the heme iron, thereby preventing turnover. MIC formation from 1 does not occur in reconstituted P450 systems with CYP2B1/2, 2C11 or 2E1, or in microsomes exposed to gentle heating to inactivate the flavin-containing monooxygenase (FMO). In contrast, N-hydroxy-N-cyclopropylbenzylamine (3) and N-benzylhydroxylamine (4) generate MICs much faster than 1 in both reconstituted and microsomal systems. MIC formation from nitrone 5 (PhCH = N(O)cPr) is somewhat faster than from 1, but very much faster than the hydrolysis of 5 to a primary hydroxylamine. Thus the major overall route from 1 to a P450 MIC complex would appear to involve FMO oxidation to 3, further oxidation by P450 and/or FMO to nitrone 5' (C2H4C = N(O)CH2Ph), hydrolysis to 4, and P450 oxidation to alpha-nitrosotoluene as the precursor to oxime 2 and the major MIC from 1.     

Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.     

真菌一氧化氮还原酶细胞色素P—450nor 2 cDNA序列的测定

真菌一氧化氮还原酶细胞色素Ｐ４５０ｎｏｒ２ｃＤＮＡ序列的测定刘德立（华中师范大学生命科学学院武汉４３００７０）ＨｉｒｏｆｕｍｉＳＨＯＵＮ（筑波大学应用生物化学系日本）在真菌的反硝化作用中,一种细胞色素Ｐ４５０起着一氧化氮还原酶的作用,被称为细胞色...     

细胞色素P450酶系循环催化的新途径

细胞色素P45 0酶系的循环催化反应需要电子供体NADPH或NADH等辅助因子系统 ,因此它在实际应用中受到制约。用电极电解或锌粉作电子供体取代NADPH辅助因子可以获得与NADPH相似的底物转换率。此外 ,还讨论了P45 0的“定向进化”产生的突变体在无NADPH等辅助因子存在下 ,通过“过氧化物途径”使底物羟基化。     

链霉菌细胞色素P450研究进展

链霉菌中存在大量的细胞色素P450,它们在链霉菌次生代谢产物的生物合成和外来化学物质代谢过程中发挥了重要作用.本文综述了链霉菌中发现的细胞色素P450及其功能的研究进展,分析了存在的问题和研究应用前景.     

Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing

Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).     

Cytochrome P450 systems—biological variations of electron transport chains

Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.     

Cytochrome P450 biosensors-a review

Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice.     

Spectroscopic features of cytochrome P450 reaction intermediates

Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2] and [3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV–vis absorption spectra of the reduced CO-saturated state [4] and [5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle.     

Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in ¹⁸O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many ¹⁸O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.