Phylogenetic Analysis of the Cytochrome P450 3 (CYP3) Gene Family

Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya. The CYP3 gene family includes the CYP3A and CYP3B subfamilies. Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates. The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds. To understand vertebrate CYP3 diversity better, we determined the killifish (Fundulus heteroclitus) CYP3A30 and CYP3A56 and the ball python (Python regius) CYP3A42 sequences. We performed phylogenetic analyses of 45 vertebrate CYP3 amino acid sequences using a Bayesian approach. Our analyses indicate that teleost, diapsid, and mammalian CYP3A genes have undergone independent diversification and that the ancestral vertebrate genome contained a single CYP3A gene. Most CYP3A diversity is the product of recent gene duplication events. There is strong support for placement of the guinea pig CYP3A genes within the rodent CYP3A diversification. The rat, mouse, and hamster CYP3A genes are mixed among several rodent CYP3A subclades, indicative of a complex history involving speciation and gene duplication. Phylogenetic analyses suggest two CYP3A gene duplication events early in rodent history, with the rat CYP3A9 and mouse Cyp3a13 clade having a sister relationship to all other rodent CYP3A genes. In primate history, the human CYP3A43 gene appears to have a sister relationship to all other known primate CYP3A genes. Other, more recent gene duplications are hypothesized to have occurred independently within the human, pig, rat, mouse, guinea pig, and fish genomes. Functional analyses suggest that gene duplication is strongly tied to acquisition of new function and that convergent evolution of CYP3A function may be frequent among independent gene copies. Current address (Rachel L. Cox): Laboratory of Aquatic Biomedicine, Marine Biology Laboratory, Woods Hole, MA 02543, USA     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

Cytochrome P450

Since 1993, three new cytochrome P450 X-ray structures have been determined, giving a total of four known structures. Two of the new structures are in the substrate-free form and one is substrate-bound. These new structures, together with a wealth of mutagenesis studies on various P450s, have provided considerable information on what structural features control substrate specificity in P450s. In addition, some important insights into the catalytic mechanism have been made.     

Phylogenetic Analysis of the NEEP21/Calcyon/P19 Family of Endocytic Proteins: Evidence for Functional Evolution in the Vertebrate CNS

Endocytosis and vesicle trafficking are required for optimal neural transmission. Yet, little is currently known about the evolution of neuronal proteins regulating these processes. Here, we report the first phylogenetic study of NEEP21, calcyon, and P19, a family of neuronal proteins implicated in synaptic receptor endocytosis and recycling, as well as in membrane protein trafficking in the somatodendritic and axonal compartments of differentiated neurons. Database searches identified orthologs for P19 and NEEP21 in bony fish, but not urochordate or invertebrate phyla. Calcyon orthologs were only retrieved from mammalian databases and distant relatives from teleost fish. In situ localization of the P19 zebrafish ortholog, and extant progenitor of the gene family, revealed a CNS specific expression pattern. Based on non-synonymous nucleotide substitution rates, the calcyon genes appear to be under less intense negative selective pressure. Indeed, a functional group II WW domain binding motif was detected in primate and human calcyon, but not in non-primate orthologs. Sequencing of the calcyon gene from 80 human subjects revealed a non-synonymous single nucleotide polymorphism that abrogated group II WW domain protein binding. Altogether, our data indicate the NEEP21/calcyon/P19 gene family emerged, and underwent two rounds of gene duplication relatively late in metazoan evolution (but early in vertebrate evolution at the latest). As functional studies suggest NEEP21 and calcyon play related, but distinct roles in regulating vesicle trafficking at synapses, and in neurons in general, we propose the family arose in chordates to support a more diverse range of synaptic and behavioral responses.     

Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements

Cytochrome P450 (CYP) enzymes play key roles in drug metabolism and adverse drug-drug interactions. Despite tremendous efforts in the past decades, essential questions regarding the function and activity of CYPs remain unanswered. Here, we used a combination of sequence-based co-evolutionary analysis and structure-based anisotropic thermal diffusion (ATD) molecular dynamics simulations to detect allosteric networks of amino acid residues and characterize their biological and molecular functions. We investigated four CYP subfamilies (CYP1A, CYP2D, CYP2C, and CYP3A) that are involved in 90% of all metabolic drug transformations and identified four amino acid interaction networks associated with specific CYP functionalities, i.e., membrane binding, heme binding, catalytic activity, and dimerization. Interestingly, we did not detect any co-evolved substrate-binding network, suggesting that substrate recognition is specific for each subfamily. Analysis of the membrane binding networks revealed that different CYP proteins adopt different membrane-bound orientations, consistent with the differing substrate preference for each isoform. The catalytic networks were associated with conservation of catalytic function among CYP isoforms, whereas the dimerization network was specific to different CYP isoforms. We further applied low-temperature ATD simulations to verify proposed allosteric sites associated with the heme-binding network and their role in regulating metabolic fate. Our approach allowed for a broad characterization of CYP properties, such as membrane interactions, catalytic mechanisms, dimerization, and linking these to groups of residues that can serve as allosteric regulators. The presented combined co-evolutionary analysis and ATD simulation approach is also generally applicable to other biological systems where allostery plays a role.     

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus

The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.     

植物细胞色素P450

对植物细胞色素P450(CYP450)基因的分离,植物CYP450在苯丙烷类物质、芥子油苷及IAA和萜类等物质的生物合成中的功能,以及对天然生物合成与人工合成物质的解毒功能等研究进展作了简要的综述。指出分离植物细胞色素P450基因,并对其生物学功能进行分析以及植物细胞色素P450降解除草剂的机制及其在环境生物修复等方面的应用是今后一段时间内植物CYP450领域的研究热点。     

Methods for Determination of Functional Activity of Cytochrome P450 Isoenzymes

The review deals with modern methods and technologies for determination of functional activity cytochrome P450 isoenzymes; these include absorbance and fluorescent spectroscopy, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), Raman, Mössbauer, and X-ray spectroscopy, surface plasmon resonance (SPR), atomic force microscopy (AFM). Methods of molecular genetic analysis have been considered in the context of personalized medicine. Using the methods of chromatography mass spectrometry it is possible to analyze reaction products formed in reactions catalyzed by cytochromes P450. Special attention is paid to modern electrochemical systems based on cytochrome P450 isoenzymes, their applicability for analysis of their catalytic activity, their use in practice and further development perspectives for experimental pharmacology, biotechnology and translational medicine.     

人细胞色素P450在大肠杆菌中的功能表达

研究人细胞色素P450（P450,CYP）在大肠杆菌中的功能表达对新药研发,临床药物治疗和药物早期ADME/T性质研究均有重要意义。异源表达人P450使用最多的宿主是大肠杆菌E.coli,然而要获得足量的有催化活性的P450仍是一个难题。结合作者近年研究,对异源表达的研究意义,P450在E.coli中功能表达的策略,高效表达的影响因素和共表达等方面做一评述,指出今后的研究应用方向。     

Camelus dromedarius Putative Cytochrome P450 Enzyme CYP2E1: Complete Coding Sequence and Phylogenetic Tree

This study determined the full-length sequence of CYP2E1, one of six cytochrome P450 genes previously examined in camel tissues by western blotting and semi-quantitative PCR. The Camelus dromedarius CYP2E1 has an open reading frame of 1,473 bp, and the cDNA encodes a protein of 490 amino acid residues with a molecular weight of 54.8 kDa. The deduced amino acid sequence showed the highest identity with Bos taurus (88%), Sus scrofa (87%), and Homo sapiens (83%). In a phylogenetic analysis, the C. dromedarius CYP2E1 isoform was located beside cattle and pigs. The deduced amino acid sequence of camel CYP2E1 showed the conserved proline-rich amino terminus and the heme-binding signature localized near the carboxy terminus of the protein.     

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.     

细胞色素P450与肿瘤

本文综棕了细胞色素Ｐ４５０同工酶与致癌物代谢、与抗癌药的相互作用以及化的关系,并对调控Ｐ４５０同工酶以防治肿瘤的策略进行了论述。由于Ｐ４５０同工酶具有多态性、工物特异性及可诱导性的特点,在调控Ｐ４５０同工酶以防治肿瘤的问题上,针对不同人群、不同疾病状况及不同用药方案可能需采取抑制或诱导的不同策略。     

Cytochrome P450s and chemoprevention

The cytochrome P450 mono-oxygenase system represents a major defence against chemical challenge from the environment, constituting part of an adaptive response mounted by an organism following exposure to harmful agents. Cytochrome P450s are also able to catalyse the activation of compounds to toxic products, and participate in a variety of essential 'housekeeping' functions, such as biosynthesis of steroid hormones and fatty acid oxidation. It is clear that the modulation of expression of these enzymes can have a significant effect on chemical toxicity, carcinogenicity and mutagenicity. The concept of cancer chemoprevention, i.e. the administration of a (non-toxic) chemical or dietary component in order to prevent neoplastic disease or to inhibit its progression, is an attractive one. Despite this, relatively little work has been done to characterize the ability of putative chemopreventive agents to modulate P450 expression, or to understand the interaction between P450s and chemopreventive agents. Before chemopreventive treatment can become a reality, it is essential that this complex issue is addressed; for instance, it is likely that any single chemopreventive agent will induce more than one P450 isoenzyme, and while altered expression of a particular P450 may attenuate the effects of one toxic agent, the effects of others might well be potentiated. Our laboratory has created a transgenic mouse line in which the rat CYP1A1 promoter drives expression of the beta-galactosidase gene. These mice can be used to define which compounds act via the Ah receptor, in which tissues, and at which stage of development. We are currently developing another mouse line in which beta1-galactosidase expression is controlled by the mouse GstA1 promoter, allowing us to define the role of the antioxidant responsive element in the action of chemopreventive agents. Finally, using cre-loxP transgenic technology, we have generated a mouse line in which P450 reductase can be deleted in a conditional, i.e. tissue-specific, manner, permitting us to investigate the role of P450s in chemoprevention in a more defined manner.     

Comparative Proteomics Among Cytochrome P450 Family 1 for Differential Substrate Specificity

Apart from playing key roles in drug metabolism and adverse drug–drug interactions, CYPs are potential drug targets to treat a variety of diseases. The intervention of over expression of P450 1A1 (CYP1A1) in tumor cells is identified as a novel strategy for anticancer therapy. We investigated three isoforms of CYP1 family (CYP1A1, CYP1A2, and CYP1B1) for their substrate specificity. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics. This can help in design of new antitumor molecule specifically metabolized by CYP1A1 to mediate their antitumor activity. In the present study, we carried out the comparative protein structure analysis of the three isoforms. Sequence alignment, root mean square deviation (RMSD) analysis, B-factor analysis was performed to give a better understanding of the macromolecular features involved in substrate specificity and to understand the interplay between protein dynamics and functions which will have important implications on rational design of anticancer drugs. We identified the differences in amino acid residues among the three isoforms of CYP1 family, which may account for differential substrate specificity. Six putative substrate recognition sequences are characterized along with the regions they form in the protein structure. Further the RMSD and B-factor analysis provides the information about the identified residues having the maximum RMSD and B-factor deviations.     

细胞色素P450与除草剂代谢

细胞色素P450是广泛存在于生物中的一类具有混合功能的血红素氧化酶。P450对除草剂代谢的机制及反应类型是多样的,与除草剂代谢相关的P450基因的植物转基因研究得到了具有不同除草剂抗性的转基因植物。文章就这方面的研究进展作介绍。     

Plant Cytochrome P450 Monooxygenases

Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxification pathways. The classic forms of these enzymes are heme-dependent mixed function oxidases that utilize NADPH or NADH and molecular oxygen to produce functionalized organic products. The nonclassical forms are monooxygenases that either do not utilize flavoproteins for dioxygen activation or fail to incorporate molecular oxygen into their final product. Biosynthetic P450s play paramount roles in the synthesis of lignin intermediates, sterols, terpenes, flavonoids, isoflavonoids, furanocoumarins, and a variety of other secondary plant products. Other catabolic P450s metabolize toxic herbicides and insecticides into nontoxic products or, conversely, activate nontoxic substances into toxic products. Biochemical and molecular characterizations on a number of plant P450s have indicated that the relationships between these heme proteins and their substrates are at least as complex as those that exist in mammalian systems. Examples now exist of plant P450s that metabolize: a narrow range of substrates to yield different products, a single substrate to yield different products, multiple substrates to yield the same product, or a single substrate sequentially to yield discrete intermediates in the biosynthesis of a single product. Extensive divergence of catalytic site as well as noncatalytic site residues accounts for the high degree of primary structure variation in the P450 gene superfamily and the diverse array of substrates synthesized and/or detoxified by these proteins. Classic P450s still retain a highly conserved F-G-R-C-G motif in their catalytic site and conserved amino acids in their oxygen binding pocket; nonclassical P450s diverge at several of these positions. A broad range of cloning and transient expression strategies are suitable for plant P450 studies and these have allowed for the isolation and characterization of a number of P450 cDNAs and genes. Because many of these sequences have been cloned only recently, much remains to be learned about the substrate specificities of P450 reactions in plants and the mechanisms by which their genes are regulated.     

Systematic Functional Study of Cytochrome P450 2D6 Promoter Polymorphisms in the Chinese Han Population

The promoter polymorphisms of drug-metabolizing genes can lead to interindividual differences in gene expression, which may result in adverse drug effects and therapeutic failure. Based on the database of CYP2D6 gene polymorphisms in the Chinese Han population established by our group, we functionally characterized the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in this population. Using site-directed mutagenesis, all the five SNPs identified and ten haplotypes with a frequency equal to or greater than 0.01 in the population were constructed on a luciferase reporter system. Dual luciferase reporter systems were used to analyze regulatory activity. The activity produced by Haplo3(−2183G>A, −1775A>G, −1589G>C, −1431C>T, −1000G>A, −678A>G), Haplo8(−2065G>A, −2058T>G, −1775A>G, −1589G>C, −1235G>A, −678A>G) and MU3(−498C>A) was 0.7−, 0.7−, 1.2− times respectively compared with the wild type in human hepatoma cell lines(p<0.05). These findings might be useful for optimizing pharmacotherapy and the design of personalized medicine.     

Cytochrome P450 2E polymorphism in feline liver

Only one isoform of cytochrome P450 (CYP) 2E subfamily was known in human and various animals. Three cDNAs corresponding to CYP 2E subfamily members (CYP2E-a, CYP2E-b and CYP2E-c) were obtained from feline liver. These cDNAs each had a 1488-bp nucleotide coding region encoding a predicted amino acid sequence of 495 residues. Eleven amino acid substitutions were observed between CYP2E-a and CYP2E-b, but only one substitution between CYP2E-b and CYP2E-c. The CO difference spectrums about 450 nm wave length and similar values of Vmax and Km of 6-hydoxygenase activity toward chlorzoxazone were observed in all three isoforms expressed in AH22 yeast cells. By PCR-RFLP, mRNA of the CYP2E-a was found to be expressed in liver, mononuclear cells, kidney, lung, stomach, intestine and pancreas, whereas CYP2E-b and CYP2E-c were expressed mainly in the liver and mononuclear cells. Expression of CYP2E-a was observed in the livers of all felines tested, but CYP2E-b and CYP2E-c were not expressed in all cats. The sequences of two different introns between exons I and II and between exons VII and VIII were obtained in genomic DNA from the feline liver. Based on these results, we conclude that cats have two highly similar CYP2E genes.