Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation.

Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the 'peroxide shunt' pathway with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.     

Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes

Jasmonate and salicylate are plant-produced signals that activate plant defence genes after herbivory or pathogen attack. Amplification of these signals, evoked by either enemy attack or experimental manipulation, leads to an increase in the synthesis of toxic compounds (allelochemicals) and defence proteins in the plants. Although the jasmonate and salicylate signal cascades activate different sets of plant defence genes, or even act antagonistically, there is substantial communication between the pathways. Jasmonate and salicylate also contribute to protecting plants against herbivores by causing plants that experience insect damage to increase their production of volatile molecules that attract natural enemies of herbivorous insects. In response to plant defences, herbivores increase their production of enzymes that detoxify allelochemicals, including cytochrome P450s (refs 15, 16). But herbivores are potentially vulnerable to toxic allelochemicals in the duration between ingesting toxins and induction of detoxification systems. Here we show that the corn earworm Helicoverpa zea uses jasmonate and salicylate to activate four of its cytochrome P450 genes that are associated with detoxification either before or concomitantly with the biosynthesis of allelochemicals. This ability to 'eavesdrop' on plant defence signals protects H. zea against toxins produced by host plants.     

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug-drug interactions.     

褐飞虱细胞色素P450基因cDNA片段的克隆、测序及表达分析

利用反转录多聚酶链式反应(RT-PCR)技术克隆了褐飞虱细胞色素P450基因编码区的cDNA片段,并进行了序列测定.结果表明,所克隆到的cDNA片段长度为237bp,经BLAST查找比对发现,该片段所编码的氨基酸序列与来自烟草天蛾、棉铃虫、埃及伊蚊、家蝇、黑腹果蝇和线虫的CYP6家族的P450的氨基酸序列存在同源性.Northern杂交分析显示,在褐飞虱取食抗性水稻后,P450基因的表达水平明显升高.以上结果表明,P450基因的表达受抗性水稻的诱导,该基因在褐飞虱对抗性水稻的耐受性和解毒方面可能起着重要作用.     

重组真菌细胞色素P450nor2在酵母细胞中的表达

酿酒酵母（Saccharomyces cerevisiae）是一种理想的真核蛋白表达系统．将真菌细胞色素P450nor2基因亚克隆到酵母表达载体pAUR123中,构建重组表达质粒pAUR-P450nor2并转化酿酒酵母AH22,经Aureobasidin A筛选和菌落PCR鉴定得到阳性克隆．SDS-PAGE分析证实：重组的真菌细胞色素P450nor2在酵母细胞中实现了高表达．     

细胞色素P450与农药相互作用及其机理研究

综述了细胞色素P450的种类和功能多样性,介绍了细胞色素P450参与除草剂代谢及其作用机理．同时,简述了细胞色素P450 14DM与杀菌剂特异性作用的机制．报道了我们从抗除草剂的瑞士黑麦草中克隆CYP8181同源基因及其功能的研究,以及柑橘绿霉菌细胞色素P450 14DM基因的克隆表达．为深入研究杀菌剂作用细胞色素P450 14DM的机理从而设计以P450 14DM为特异性靶标的新型农药、以及进一步研究细胞色素P450酶系与除草剂代谢的关系打下了良好的基础．     

Metabolism of fibrates by cytochrome P450s and UDP-glycosyltransferases in rat and human liver microsomes

Fibrates are widely used for the treatment of dyslipidemia.However,the contributions of the phase I and phase II metabolic pathways to the clearance of fibrates are unclear.In this study,we investigated the metabolism of gemfibrozil(Gem) ,clofibric acid(CA) ,fenofibric acid(FA) and bezafibrate(Beza) by cytochrome P450s(P450s) and UDP-glycosyltransferases(UGTs) using a substrate depletion approach.We also compared the metabolic characteristics of rat liver microsomes(RLM) and human liver microsomes(HLM) .The intrinsic clearance rates mediated by P450s,UGTs and both were 172 ± 22,643 ± 26,798 ± 103 μL min-1 mg-1,respectively,for Gem and 43 ± 11,88 ± 12,119 ± 15 μL min-1 mg-1,respectively,for CA in RLM.The fractions metabolized by P450s and UGTs in RLM were 22% and 81% for Gem,36% and 74% for CA.The P450-and UGT-mediated depletion rates for Gem were 303 and 1607 nmol min-1 mg-1 in RLM versus 86 and 243 nmol min-1 mg-1 in HLM.The corre-sponding rates for CA were 1.1 and 1.7 nmol min-1 mg-1 in RLM versus 0.025 and 0.038 nmol min-1 mg-1 in HLM.Accordingly,both P450s and UGTs substantially contribute to the clearance of Gem and CA,with UGTs playing a greater role.To avoid un-der-estimating the impact of these pathways,it is necessary to measure NADPH-and UDPGA-dependent metabolism.Although the fractions of these two pathways in RLM and HLM were similar,the depletion rate of Gem and CA in RLM was higher than that in HLM.The metabolism of FA and Beza by P450s and UGTs was too low to calculate intrinsic clearance in both RLM and HLM.These results indicate that fibrates are metabolized via similar pathways in rats and humans,and it is applicable to use RLM to predict the clearance of fibrates in human.     

3个白桦细胞色素P450基因生物信息学及表达分析

【目的】研究白桦细胞色素P450基因表达的组织特异性,以及在茉莉酸甲酯(MeJA)、水杨酸(SA)、赤霉素(GA₃)、脱落酸(ABA)、乙烯利和伤害处理下的基因表达模式。【方法】筛选白桦转录组,获得3个BpCYP450 基因,分别命名为BpCYP4、BpCYP5、 BpCYP14,利用生物信息学分析BpCYP450蛋白的分子结构特征及其与其他物种CYP450蛋白的亲缘关系。采用QRT-PCR技术,对白桦BpCYP450组织特异性、激素信号及伤害诱导下的表达特征进行分析。【结果】生物信息学结果表明BpCYP4、BpCYP5、BpCYP14 cDNA序列长度分别为1 569、1 584和1 530 bp,具有完整的开放阅读框(ORF),分别编码522、527和509个氨基酸,均为亲水性跨膜蛋白,主要定位于叶绿体。白桦BpCYP450与百脉根、扁豆及豌豆等豆科植物CYP450蛋白亲缘性较高。组织特异性分析结果显示,3个BpCYP450 基因在叶和根中表达较高,茎中较低。激素信号及伤害诱导结果表明,3个BpCYP450基因不同程度地响应MeJA、SA、GA₃、ABA、乙烯利激素信号及伤害诱导。【结论】3个BpCYP450基因均为CYP450基因家族的新成员,具有组织特异性及激素诱导表达特性,可能在白桦生长发育、抵御胁迫及代谢物合成中发挥重要作用。     

Identification of the primary gene defect at the cytochrome P450 CYP2D locus

The mammalian cytochrome P450-dependent monooxygenase system is involved in the metabolism of drugs and chemical carcinogens. The role of these enzymes in toxicological response is exemplified by an autosomal recessive polymorphism at the cytochrome P450 CYP2D6 debrisoquine hydroxylase locus which results in the severely compromised metabolism of at least 25 drugs, and which in some cases can lead to life-threatening side-effects. In addition, this polymorphism, which affects 8-10% of the caucasian population, has been associated with altered susceptibility to lung and bladder cancer. Here we report the identification of the primary mutation responsible for this metabolic defect and the development of a simple DNA-based genetic assay to allow both the identification of most individuals at risk of drug side-effects and clarification of the conflicting reports on the association of this polymorphism with cancer susceptibility.     

诺氟沙星对水生生物细胞色素P450作用机制研究进展

环境中的抗生素主要来源于生活污水、医疗废水以及水产养殖废水等.2013年中国抗生素总使用量约为1.62×10⁵ t,其中48%为人用抗生素,其余为兽用抗生素.大量具有活性的抗生素经地表径流和城市排污管道最终积聚到河流湖泊中.这些活性物质会对水生生物产生危害,尤其是在较长的时间跨度上,这种危害会进一步扩大.文章概述了近几年基于细胞色素P450(cytochrome P450,CYP450)作为检测指标,诺氟沙星(norfloxacin,NFLX)对一些水生生物的生态毒理效应.同时介绍了NFLX对于选取的指标CYP450的影响机制,以及检测这些指标的可行性方法.     

Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue

As a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P45015 alpha and P450coh, for example, which are expressed in female liver and male kidney cells, catalyse 15 alpha-hydroxylation of delta 4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues. To determine the structural basis of the different substrate specificities of P45015 alpha and P450coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.     

Clustered spikelets 4, encoding a putative cytochrome P450 protein CYP724B1, is essential for rice panicle development

Rice （Oryza sativa L.） inflorescence （panicle） architecture is an important agronomic trait, serving as one of the determinants of rice yield. A number of genes related to panicle development have been cloned and functionally characterized so far. However, more information is needed for fully understanding of the mechanism underlying the panicle development. In the present study, we identified a clustered spikelets 4 （cl4） mutant in the 93-11 genetic background. Compared to its wild-type 93-11, cl4 mutant has a typical clustered spikelets phenotype with all primary branches clustered on the base of the main rachis and 2-3 abnormal spikelets clustered on the primary branches. Moreover, cl4 mutant also shows shorter plant height than that of the wild type. Map-based cloning strategy is per- formed to clone the CL4 gene. As a result, CL4 is demonstrated to encode a putative cytochrome P450 protein CYP724B1, which is involved in brassinosteroid biosynthesis. To confirm our mapping result, the CL4 RNAi transgenic plants are generated. And the transgenic plants also show similar phenotype as the cl4 mutant. These results provide strong evidence that CL4 plays an important role in rice panicle development as well as plant height regulation.     

ConformationsofCytochromeP─450Modelsby1Dand2DNMR

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重组家蝇细胞色素P450 6A1的大肠杆菌对艾氏剂的生物降解作用

以大肠杆菌为宿主表达家蝇细胞色素P450 6A1(CYP6A1)酶,在CYP6A1基因后面加入人细胞色素P450还原酶CPR基因,验证了CPR酶对CYP6A1酶催化功能的影响,和CYP6A1酶、重组大肠杆菌对艾氏剂的代谢能力.结果表明:CYP6A1蛋白对艾氏剂有环氧化作用,在CYP6A1对艾氏剂催化中起关键作用.将5μmol/L艾氏剂加入重组大肠杆菌菌液8 h可检测到163.36nmol/L艾氏剂环氧化产物狄氏剂,说明CYP6A1-CPR宿主菌对艾氏剂具有代谢能力,可用于艾氏剂污染的土壤的生物降解.     

Up-regulation of endothelial nitric oxide synthase by cytochrome P450 arachidonic acid epoxygenase BM3F87V

Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), have been suggested to be an endothelium-derived hyperpolarizing factor (EDHF). However, the interaction or relation between EDHF and endothelial nitric oxide synthase (eNOS) is still to be elucidated. In the present study, the regulation of eNOS by endogenous EDHF is examined. The cytochrome P450 epoxygenase BM3F87V is cloned into the mammalian expression vector pCB6. Cultured bovine aortic endothelial cells (BAECs) less than 4 passages are used and transfected with BM3F87V. The effects of endogenous EETs result from BM3F87V transfection on eNOS are assessed in the endothelial cells by Western blot and Northern blot, and eNOS activity is also measured by the conversion of L-arginine to L-citrulline. Compared to transfection with the empty pCB6 vector, transfection of BAECs with BM3F87V significantly elevates the levels of eNOS protein expression, which is markedly inhibited by treatment with CYP inhibitor 17-ODYA. BM3F87V transfection also elevates the eNOS mRNA level and increases the eNOS activity. This study suggests that EDHF up-regulates eNOS gene expression.     

家蚕基因组P450基因家族的分析

利用家蚕基因组mRNA和氨基酸数据库对P450基因进行搜索．结果显示：在家蚕基因组中发现77个P450基因．分别归于18个P450家族和26个亚家族,其中有9个多基因家族。3个基因家族的成员超过10个,CYP4有20个成员。是一个典型的多基因家族。对P450基因进行正选择、基因转换和基序分析。结果表明：1个组（包含3个P450s基因）中检测出了显著的正选择替换位点,在这1个组中．转换事件的次数为3次。     

大黄苷元对大鼠药物代谢酶CYP2A6、CYP3A4活性的影响

研究大黄苷元对大鼠体内药物代谢酶CYP2A6、CYP3A4活性的影响,以预测大黄与临床常用药物的相互作用.大鼠分组,分别灌胃不同质量浓度大黄苷元、质量分数0.5%羧甲基纤维素钠(CMC-Na)、苯巴比妥钠(PB)、地塞米松(DEX)、β-奈黄酮(β-NF)和酮康唑(KET),取各给药组给药后24 h的肝微粒体(RLM)与CYP2A6、CYP3A4的探针底物香豆素、睾酮进行体外温孵,通过高效液相色谱法分别测定各底物的代谢产物7-羟基香豆素、6β-羟基睾酮的生成量,考察大黄苷元对CYP2A6、CYP3A4活性的影响.大黄苷元高、中、低剂量给药组代谢产物7-羟基香豆素的生成量高于空白给药组和KET给药组,均有统计学意义(P0.05)、(P0.01),大黄苷元高剂量给药组代谢产物6β-羟基睾酮的生成量高于空白给药组和KET给药组,均有统计学意义(P0.05)、(P0.01).随着大黄苷元给药剂量的增加,7-羟基香豆素、6β-羟基睾酮的生成量均随之增加.大黄苷元对CYP2A6、CYP3A4均有诱导作用,并且随着大黄苷元剂量的增加,其对CYP2A6、CYP3A4诱导作用均增强.     

尖镰孢菌细胞色素P45055A1与NO相互作用的荧光光谱法研究

以大肠杆菌为宿主表达了尖镰孢菌细胞色素P450 55A1(CYP450 55A1),并采用荧光光谱法研究了其与NO的相互作用.结果表明:在大肠杆菌(DE3)中成功表达了具有酶活性的CYP55A1.在280 nm波长激发下,NO的加入使CYP55A1的荧光逐渐降低,但随温度的变化差异较小;在413 nm波长激发下,NO的加入使CYP55A1的荧光逐渐升高,但加入NADH后其荧光又恢复到原来的值.     

磷掺杂半导体单壁碳纳米管电子结构的第一性原理研究

采用基于密度泛函理论的第一性原理,研究了掺磷(P)单壁碳纳米管(SWCNT)的电子结构性质.结果表明,引入掺杂原子可显著改变SWCNT费米能级附近的能带结构,掺杂SWCNT的电子态密度(DOS)向低能端移动,其最高分子占据轨道(HOMO)与最低分子非占据轨道(LUMO)间的能隙减小,掺杂的磷原子比碳原子多出的电子更容易从价带向导带跃迁.