细胞色素P450的生物多样性和植物保护

细胞色素P450单加氧酶能催化一系列内源性或外源性亲脂化合物的氧化反应。该酶系统分成三个结构区域:黄素腺嘌呤二核昔酸(FAD)、Fe-S或黄素单核苷酸(FMN)和血红素。FAD和Fe—S/FMN部分构成了从还原型辅酶Ⅱ或还原型辅酶Ⅰ到对氧化反应起催化作用的血红素部分的电子传递链。在原核生物和真核生物之间这些区域结构由1、2或3个蛋白质组成。在原核生物细胞中,细胞色素P450酶系主要有三个组成部分;细胞色素P450(P450)、铁     

HPLC法同时检测大鼠血浆中3种细胞色素P450探针药物

目的:建立同时检测大鼠血浆中3种细胞色素P450(CYP450)探针药物,即氯唑沙宗(CYP2E1)、甲苯磺丁脲(CYP2C9)和咪达唑仑(CYP3A4)的高效液相色谱方法。方法:色谱条件选用Topsil TM C_(18)column(250mm×4.6mm,5μm);流动相为甲醇-(NH_4)_2HPO_4(54∶46,p H值为3.4);检测波长230nm;流速1.0m L·min~(-1);柱温30℃。结果:氯唑沙宗、咪达唑仑、甲苯磺丁脲的线性范围分别为0.2～50,0.2～50,0.1～50μg·m L~(-1);方法回收率分别为96.22%～101.42%,98.72%～104.14%,97.09%～105.27%。结论:该HPLC检测方法快捷,准确,灵敏度高,符合生物样品分析要求,适用于同时测定3种CYP450酶亚型探针药物的大鼠血浆药物浓度,可为相关肝药酶代谢研究提供方法学参考。     

细胞色素P450酶系对除草剂代谢作用的研究进展

细胞色素P450酶系是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。在高等植物中执行着各种不同功能．包括植物正常生长发育所必需的初级代谢物和次级代谢物的生物合成，而且参与许多外源性物质包括除草剂等的生物氧化。笔者综述了与除草剂代谢有关的细菌、哺乳动物和植物细胞色素P450酶系，概述了细胞色素P450酶系代谢作用在除草剂选择性、耐受性、抗药性机理和抗除草剂作物品种培育方面的意义，以及转细胞色素P450基因风险性评价等。     

细胞色素P450酶代谢物的先导物研发

细胞色素P450(CYP)酶在药物代谢方面发挥着重要作用,它可以催化一些有机反应,生成一些具有新型药理活性的化学实体。本文主要从CYP参与大量天然产物的生物合成途径,及一些已知药物通过CYP参与的代谢后生成药理活性物质两个方面,介绍细胞色素P450酶代谢物的研发现状。     

共表达口蹄疫病毒O型P1-2A-IL-18基因和Asia I型P1-2A-3C基因重组鸡痘病毒的构建

目的构建共表达口蹄疫病毒(FWDV)O型P1-2A-IL-18基因和AsiaI型P1-2A-3C基因的重组鸡痘病毒。方法将酶切得到的FWDVO型P1-2A-IL-18基因和AsiaI型P1-2A-3C基因克隆至鸡痘病毒表达载体pUTAL上,构建重组鸡痘病毒转移质粒pUTAL-P1-2A-3C-P1-2A-IL-18,与鸡痘病毒(FPV)282E4株共转染鸡胚成纤维细胞(CEF),通过BrdU3次加压筛选,挑选出单克隆重组病毒株,进行RT-PCR及间接免疫荧光(IFA)鉴定。结果重组鸡痘病毒转移质粒pUTAL-P1-2A-3C-P1-2A-IL-18经双酶切鉴定证明构建正确。经RT-PCR和IFA鉴定,证明所筛选的重组鸡痘病毒在CEF中能正确表达P1-2A-3C-P1-2A-IL-18基因盒。结论已成功构建了共表达FMDVO型P1-2A-IL-18基因和AsiaI型P1-2A-3C基因的重组鸡痘病毒。     

葡萄柚中细胞色素P450酶抑制剂的研究进展

葡萄柚果汁中富含呋喃香豆素类化学成分,这些呋喃香豆素类化合物特别像6,7-DHB及其类似物能抑制药物代谢酶细胞色素P450,以致于提高特定药物口服生物利用率或阻碍致癌物质的活性。综述了近年来发现的呋喃香豆素化合物的类别、活性及合成方面的情况。     

定点突变提高细胞色素P450 BM-3吲哚羟基化能力

为进一步提高细胞色素P450 BM-3(A74G/F87V/L188Q)对吲哚的羟基化能力,根据酶结构与功能的关系,以突变酶E435T为基础,在168位点引入D168L突变,获得了吲哚羟基化能力得到显著提高的突变酶D168L/E435T。突变酶对吲哚的K_m为1.72 mmol·L^-1(父本2.09 mmol·L^-1),转化数(k_cat)为28.15 min^-1(父本4.04 min^-1),表明D168L定点突变可以略微提高酶对底物的亲和力,但主要的效应是促进了底物的转化速率,这两个效应的综合表现是使酶的催化效率(k_cat K_m^-1)比父本酶提高了8.48倍。此外,产物中副产物靛玉红的比例也降低为1.2%(父本7.3%),这说明该突变酶催化吲哚的区域选择性上也更有利于靛蓝的生成。     

细胞色素P450 BM-3羟基化吲哚能力的半理性改造

为进一步改造细胞色素P450 BM-3酶对吲哚的羟基化能力,以P450 BM-3结构与功能关系的推测为指导,选择突变酶P450 BM-3 （A74G/F87V/L188Q/E435T）为父本,在可能影响P450 BM-3催化吲哚羟基化区域选择性的D168位点进行定点饱和突变,根据全细胞催化产物颜色及组成进行筛选,得到了产物组成、酶动力学性质与父本不同的两个突变酶。突变酶D168W的吲哚羟基化产物中90%是靛玉红,而另一个突变酶D168R的产物中87%是靛蓝,产物组成均不同于亲本。在催化吲哚羟基化时,D168W的k_cat与父本相当,但K_m却是父本的4.8倍,催化活力只有父本的20%;而D168R的k_cat是父本的1.9倍,K_m是父本的82%,催化活力比父本提高了1.37倍。结果表明,在E435T突变上叠加D168位氨基酸残基突变对酶的催化性质产生了单一位点突变所不具有的协同效应,对酶催化的区域选择性和催化活力都有显著影响,以致改变了催化产物组成。这种基于知识的半理性定向进化方法由于是在关键位点进行突变,因此突变目的性强、突变效果显著。     

金属蛋白的配位化学：细胞色素P450研究进展

介绍了细胞色素P_(450)的研究进展。     

氨基酸突变对细胞色素P450 BM-3羟基化吲哚能力的影响

利用饱和定点突变技术对细胞色素P450 BM-3（A74G/F87V/L188Q）（PT）的168和435氨基酸位点分别进行随机突变,根据其羟基化吲哚生成的靛蓝在670nm处具有特殊吸收峰进行高通量筛选,获得了三个高于亲本酶（PT）的突变酶D168H、D168L和E435T。通过考察突变酶反应动力学参数、电子耦合率、热稳定性、最适pH以及区域专一性的变化情况,发现相对亲本酶而言所有的突变酶对底物吲哚的亲和力和催化效率都得到了不同程度的提高,其中突变酶D168H的kcat值比亲本酶提高了3倍多;突变酶D168H的电子耦合率比亲本酶大约降低了2倍,而突变酶D168L和E435T的电子耦合率却有轻微的提高;所有突变酶均在pH8.2附近表现出最大羟基化吲哚生产靛蓝的能力;突变酶D168H对吲哚的区域选择性得到了提高,主产物靛蓝从亲本酶的72%提高到了93%。另外,热稳定性实验表明：尽管突变酶D168H和D168L的热稳定性较亲本酶有所降低,但活力的大幅度提高并未对酶的热稳定性造成大的伤害。以上所有的结果为了解细胞色素P450 BM-3结构与功能关系提供了有用的信息,同时为进一步定向进化P450BM-3提高其功能特性提供了进化模板。     

Phytanic acid α-hydroxylation by bacterial cytochrome P450

Fatty acid α-hydroxylase, a cytochrome P450 enzyme, from Sphingomonas paucimobilis, utilizes various straight-chain fatty acids as substrates. We investigated whether a recombinant fatty acid α-hydroxylase is able to metabolize phytanic acid, a methyl-branched fatty acid. When phytanic acid was incubated with the recombinant enzyme in the presence of H₂O₂, a reaction product was detected by gas chromatography, whereas a reaction product was not detected in the absence of H₂O₂. When a heat-inactivated enzyme was used, a reaction product was not detected with any concentration of H₂O₂. Analysis of the methylated product by gas chromatography-mass spectrometry revealed a fragmentation pattern of 2-hydroxyphytanic acid methyl ester. By single-ion monitoring, the mass ion and the characteristic fragmentation ions of 2-hydroxyphytanic acid methyl ester were detected at the retention time corresponding to the time of the product observed on the gas chromatogram. The K _m value for phytanic acid was approximately 50 μM, which was similar to that for myristic acid, although the calculated V _max for phytanic acid was about 15-fold lower than that for myristic acid. These results indicate that a bacterial cytochrome P450 is able to oxidize phytanic acid to form 2-hydroxyphytanic acid.     

Structure-activity correlations in pentachlorobenzene oxidation by engineered cytochrome P450cam

We had reported engineering of the heme monooxygenase cytochrome P450cam from Pseudomonas putida with the F87W/Y96F/L244A/V247L mutations for the oxidation of pentachlorobenzene (PeCB), a recalcitrant environmental contaminant, to pentachlorophenol. In order to provide further insights into P450 structure, function and substrate recognition, we have determined the crystal structure of this 4-mutant without a substrate and its complex with PeCB. PeCB is bound face-on to the heme, with a weak Fe--Cl interaction. One PeCB chlorine is located in the cavity generated by the L244A mutation, in striking illustration of the role of this mutation in promoting PeCB binding. The structures also show that the P450(cam) oxygen-binding groove between G248 and T252 is flexible and can tolerate significant deviations from their conformations in the wild type without loss of enzyme activity. Analysis of the PeCB binding interactions led to introduction of the T101A mutation to enable the substrate to reorient during the catalytic cycle for more efficient oxidation. The resultant 5-mutant F87W/Y96F/T101A/L244A/V247L is 3-fold more active for PeCB oxidation than the 4-mutant. Polychlorinated benzene binding by the mutants and the partitioning between substrate oxidation and non-productive (uncoupling) side reactions are correlated with the structural data.     

Bisallylic hydroxylation and epoxidation of polyunsaturated fatty acids by cytochrome P450

Polyunsaturated fatty acids can be oxygenated by cytochrome P450 to hydroxy and epoxy fatty acids. Two major classes of hydroxy fatty acids are formed by hydroxylation of the ω-side chain and by hydroxylation of bisallylic methylene carbons. Bisallylic cytochrome P450-hydroxylases transform linoleic acid to 11-hydroxylinoleic acid, arachidonic acid to 13-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid, 10-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and 7-hydroxyeicosa-5Z,8Z,11Z,14Z-tetraenoic acid and eicosapentaenoic acid to 16-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid, 13-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid and 10-hydroxyeicosa-5Z,8Z,11Z,14Z,17Z-pentaenoic acid as major metabolites. The bisallylic hydroxy fatty acids are chemically unstable and decompose rapidly tocis-trans conjugated hydroxy fatty acids during acidic extractive isolation. Bisallylic hydroxylase activity appears to be augmented in microsomes induced by the synthetic glucocorticoid dexamethasone and by some other agents, but the P450 gene families of these hydroxylases have yet to be determined. The fatty acid epoxides, which are formed by cytochrome P450, are chemically stable, but are hydrolyzed to diols by soluble epoxide hydrolases. Epoxidation of polyunsaturated fatty acids is a prominent pathway of metabolism int he liver and the renal cortex and epoxygenase activity appears to be under homeostatic control in the kidney. Many arachidonate epoxygenases have been identified belonging to the CYP2C gene subfamily. Epoxygenases have also been found in the central nervous system, endocrine organs, the heart and endothelial cells. Epoxides of arachidonic acid have been found to exert pharmacological effects on many cells.     

Hypersensitive radical probes and the mechanisms of cytochrome P450-catalyzed hydroxylation reactions

The title probes are precursors to kinetically calibrated, aryl-substituted cyclopropylcarbinyl radicals that rearrange with picosecond lifetimes. Applications in studies of cytochrome P450-catalyzed hydroxylation reactions are reviewed. Initially confusing results regarding lifetimes of radicals in the hydroxylation reactions were resolved when second-generation probes that distinguish between radicals and cations were employed. The results indicate that two electrophilic oxidizing species are involved in P450-catalyzed hydroxylations, an iron-oxo species that inserts oxygen and a hydroperoxo-iron species that inserts OH(+). The cationic rearrangement products are ascribed to reactions of the protonated alcohol products formed from the latter.