细胞色素P450介导的昆虫抗药性

本文介绍了昆虫细胞色素P450(简称P450)及其介导抗性的分子基础的研究进展。细胞色素：P450在转录水平上的过量表达是P450介导抗性的主要机制,P450的氨基酸残基改变也可能改变昆虫的抗药性。     

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.     

细胞色素P450介导的果蝇抗药性研究进展

细胞色素P450 (cytochrome P450, CYP450)超基因家族是由一些数量多而功能复杂的血红蛋白酶基因所组成,该代谢酶系作为一种几乎地球上所有需氧生物都存在的重要生存策略,可以调控多种内源物质及外源化合物的代谢,参与了众多重要的生命过程,代谢解毒作用是该酶系重要功能之一。细胞色素P450的代谢解毒作用受药物影响,机体通过改变基因表达量,实现增强代谢解毒,加快机体对于有害物质的代谢,从而使得机体对有害环境产生一定的适应性,进而使得机体产生耐药性或抗药性。本研究说明果蝇细胞色素P450介导的杀虫剂类药物代谢机制及代谢抗性的特点等方面的研究,对明确果蝇的抗药性机制研究具有参考意义。     

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。     

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.     

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.     

Cytochrome P450 Genes from the Sacred Lotus Genome

Cytochrome P450 monooxygenases (P450s) in the sacred lotus (Nelumbo nucifera) genome have been identified and named according to systematic P450 nomenclatures. Comparisons of these sequences with those in the papaya and grape CYPomes have indicated that gene blooms exist in the CYP89, CYP94, CYP96 and CYP714 families and that less dramatic expansions exist in the CYP71 and CYP72 families. Expansions in the CYP94 and CYP96 families may be associated with generation of the extremely hydrophobic leaf surfaces associated with the “lotus effect” in this water-adapted species, since these families are known to hydroxylate fatty acids and alkanes in the wax biosynthetic pathways of other plant species. Evolution of the CYP719 and CYP80 families may be associated with production of a number of benzylisoquinoline and aporphine alkaloids. Structures for anonaine and roemerine, two of the most abundant aporphine alkaloids in lotus leaves and seeds, contain methylenedioxy bridges that are known to be generated by members of the CYP719 family. With only one CYP719A22 gene existing in the lotus genome, it is likely that it is involved in making aporphine alkaloids. The fact that CYP719 has not previously been seen in angiosperm phylogeny below the order of Ranunculales suggests that its presence in lotus (in the Proteales) presents an evolutionary terminus prior to its loss in more recent eudicot species. With several CYP80 family genes existing in the lotus genome, there are multiple candidates for those involved in conducting benzylisoquinoline alkaloid synthesis.     

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.     

Cytochrome P450 in adrenocortical mitochondria

Summary Cytochrome P₄₅₀ in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P₄₅₀ each catalyzing a specified reaction have been purified to a homogeneous state, that is, P_450scc having cholesterol side chain cleavage activity and P₄₅₀₁₁ having 11-hydroxylation activity. The properties of these purified P₄₅₀'s as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P₄₅₀ in the mitochondria) preparation in situ.Among many findings, both purified cytochromes P₄₅₀ were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P₄₅₀. These effects are important from the point of view that the primary role of the heme of cytochrome P₄₅₀ is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P₄₅₀ in the reconstituted system have also been described Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method.     

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。     

Cytochrome P450 pattern revision.

The pattern suggested for the structure-function superfamily of cytochromes P450 is composed by combining the conserved amino acid motifs. The sizes of P450 cytochromes were estimated according to their length. The empirical coefficients reflecting the peculiarities of the primary structure of these enzymes are calculated. We propose an approach for determining novel proteins sequences to the mentioned superfamily on the ground of the complex of these parameters. A number of the hypothetical proteins from the international databases is related to the cytochromes P450 by means of our pattern.     

Cytochrome P450 52A3: Modelling of 3D Structure and Surface Mutations

Abstract

Earlier W.-H. Schunck et al. [1] have prepared a water soluble enzymatically active fragment of cytochrome P450 52A3 (CYP52A3) which is lack of 66 amino acid residues, existed as a dimer in aqueous solution. Now we propose 3D structure of the fragment, which is based on multiple sequence alignment of the CYP52A3 with its homologues proteins of known 3D structure: CYP101, 102, 107A1 and 108. The structural model have been optimised and used as a prototype for computer simulation of point mutations. These mutations should bring some changes in the surface properties, interfering dimer formation. For this aim the point of 22 hydrophobic amino acid residues have been sequentially replaced with that of charged amino acids (GLU, ASP, ARG and LYS). The scoring of “mutants” was conducted based on the changes of protein surface hydrophobicity and protein-solvent interaction energy. An analysis of the surface hydrophobicity and protein-solvent interactions permit to select most sensitive three sites (171, 352 and particularly 164 amino acid residues). The dimerization of the following “mutant” fragments must be investigated experimentally.     

细胞色素P450 3A7羟化维生素D3的功能研究

本研究通过体外生化实验研究细胞色素P450 3A7对维生素D3的羟化作用。根据GenBank报道的序列设计特异引物,扩增cyp3a7的编码区,将cyp3a7的编码区插入到pcDNATM3.1/myc-His(-) A的XhoⅠ/Bam HⅠ,通过测序检测序列的正确性。pcDNA-CYP3A7及pcDNA分别瞬时转染293T细胞,48 h后收集细胞,提取S9组分,用Bradford法测定蛋白质浓度。S9组分经12%SDS-PAGE凝胶电泳和Western blotting检测,用myc抗体作为一抗检测CYP3A7在293T细胞的表达水平。0.6 mg S9组分与1μmol/L维生素D3于37℃孵育30 min,用4倍体积的氯仿甲醇(体积比为3∶1)抽提,有机相在氮气流下吹干,残基用于HPLC分析。结果显示,重组表达CYP3A7的293T细胞的S9组分通过Western blotting检测到了特异的约60 kD的条带,对照样品未检测到特异条带的蛋白质。重组表达CYP3A7的293T细胞S9组分的孵育样品通过HPLC检测到了25-羟基维生素D3,对照样品未检测到25-羟基维生素D3。结果表明重组表达的CYP3A7羟化维生素D3生成25-羟基维生素D3。本研究为进一步探究还有哪些P450参与维生素D3在鸡体内的代谢,为阐明其代谢途径提供理论依据。     

Cytochrome P450 reductase from Candida apicola: versatile redox partner for bacterial P450s

Candida apicola belongs to a group of yeasts producing surface-active glycolipids consisting of sophorose and long-chain (ω)- or (ω-1)-hydroxy fatty acids. Hydroxylation of the fatty acids in this strain is likely catalyzed by cytochrome P450 monooxygenases (P450), which require reducing equivalents delivered via a cytochrome P450-diflavin reductase (CPR). We herein report cloning and characterization of the cpr gene from C. apicola ATCC 96134. The gene encoding a protein of 687 amino acids was cloned in Escherichia coli and the enzyme was expressed in functional form after truncation of its N-terminal putative membrane anchor. The truncated recombinant protein showed cytochrome c reducing activity (K _M of 13.8 μM and k _cat of 1,915 per minute). Furthermore, we herein demonstrate to our best knowledge for the first time the use of a eukaryotic CPR to transfer electrons to bacterial P450s (namely CYP109B1 and CYP154E1). Cloning and characterization of this CPR therefore is not only an important step in the study of the P450 systems of C. apicola, but also provides a versatile redox partner for the characterization of other bacterial P450s with appealing biotechnological potential. The GenBank accession number of the sequence described in this article is JQ015264.     

Comparison of Cytochrome P450 Genes from Six Plant Genomes

Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms, CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families, of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387 P450 sequences have been identified and annotated in plant databases. As no new angiosperm CYP families have been discovered since 2004, it is now apparent that the sampling of CYP diversity is beginning to plateau. This review presents a comparison of 1,415 cytochrome P450 sequences from the six sequenced genomes of Vitis vinifera (grape), Carica papaya (papaya), Populus trichocarpa (poplar), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis or mouse ear’s cress) and Physcomitrella patens (moss). An evolutionary analysis is presented that tracks land plant P450 innovation over time from the most ancient and conserved sequences to the newest dicot-specific families. The earliest or oldest P450 families are devoted to the essential biochemistries of sterol and carotenoid synthesis. The next evolutionary radiation of P450 families appears to mediate crucial adaptations to a land environment. And, the newest CYP families appear to have driven the diversity of angiosperms in mediating the synthesis of pigments, odorants, flavors and order-/genus-specific secondary metabolites. Family-by-family comparisons allow the visualization of plant genome plasticity by whole genome duplications and massive gene family expansions via tandem duplications. Molecular evidence of human domestication is quite apparent in the repeated P450 gene duplications occurring in the grape genome.     

棉铃虫细胞色素P450的分子生物学

棉铃虫 [Ｈｅｌｉｃｏｖｅｒｐａａｒｍｉｇｅｒａ(ｚｅａ) ]属鳞翅目夜蛾科 ,是一种寄主范围广、危害严重的世界性农业害虫。由于长期以来主要依靠化学防治 ,棉铃虫已对拟除虫菊酯、有机磷、氨基甲酸酯等多种类型的农药产生不同程度的抗性 ,给工农业生产带来巨大损失。对棉铃虫抗性机制研究发现 ,细胞色素Ｐ45 0酶系 (以下简称Ｐ45 0酶系 )代谢活性的增强是一个很重要的原因。作为Ｐ45 0酶系的重要组分 ,细胞色素Ｐ45 0 (简称Ｐ45 0 )是一类由基因超家族编码的同工酶。由于难以从昆虫体内分离出单一型Ｐ45 0 ,用传统的酶学和代谢方法很难对…     

细胞色素P450BM3催化正十六烷动力学计算

细胞色素P450 BM3作为烷烃羟基化酶,能催化正链烷烃,已被广发研究和应用.利用动力学模拟软件对BM3酶与烷烃底物复合物进行构象、酶的活性位点以及结合能的预测,并通过模拟水以及离子环境下对复合物的影响,从能量及构象位移的角度阐述BM3酶与底物结合的机理,从而用分子动力学观点来解释细胞色素P450催化烷烃机理.用Auto dock等软件将BM3与十六烷对接,发现底物C16与铁原子间距为7.57 ?,并发现与底物结合的活性位点关键残基:ALA 330,ALA 74,SER 72,GLN 73,ALA328,LEU 188,LEU 437.经Gromacs动力学模拟步长为1 ns,温度在298 K,压力为常压1.0,复合物结合稳定.