二氢乳清酸脱氢酶的结构特征和催化循环研究进展

二氢乳清酸脱氢酶是黄素依赖的线粒体酶,它催化嘧啶从头合成的第4步反应,将二氢乳清酸氧化为乳清酸.通过选择性抑制二氢乳清酸脱氢酶,从而抑制嘧啶的合成,已被开发用于治疗癌症、自身免疫性疾病、细菌或病毒感染以及寄生虫疾病等.抑制剂的开发需详细了解二氢乳清酸脱氢酶的结构特征和催化循环机制.因此,文中主要从这两个方面进行了综述,...     

微生物来源的二氢乳清酸脱氢酶抑制剂F01WB-1315A，B

摘要：目的 从微生物次生代谢产物中筛选免疫相关疾病治疗药物重要靶点—－二氢乳清酸脱氢酶的抑制剂。方法 利用自建的快速、高效的二氢乳清酸脱氢酶抑制剂的高通量筛选方法,从4560株真菌菌株中筛选阳性菌株。阳性菌株的发酵产物进行分离纯化获得活性化合物,再通过对活性化合物的紫外、质谱、核磁等理化数据的分析进行结构鉴定。结果 筛选分离得到2个活性化合物F01WB-1315A和F01WB-1315B。F01WB-1315A对二氢乳清酸脱氢酶有强的抑制活性,IC50=0.07 μg/mL,于20 μg/mL浓度下对体外     

人二氢乳清酸脱氢酶蛋白的表达、纯化及其与新型抑制剂的晶体结构

人二氢乳清酸脱氢酶（human dihydroorotate dehydrogenase, hDHODH）是催化嘧啶从头合成途径的一个关键酶。近年来,多种研究表明,抑制该酶可缓解类风湿性关节炎的症状。但该酶的抑制剂甚少,寻找该酶的高效抑制剂具有重要意义。本研究利用PCR技术扩增hDHODH基因,构建重组质粒pET-19b-hDHODH,并在大肠杆菌(Escherichia coli, E.coli ) BL21(DE3)中表达,获得可溶性蛋白质。用Ni2+-NTA亲和层析柱对蛋白质进行纯化,获得较高（90%）纯度的hDHODH蛋白,将蛋白质与抑制剂3-(5-乙硫基)-1H-1, 2, 4-三氮唑-3-)苯甲酸和底物DHO混合孵育。用Hampton试剂盒初筛晶体并用棋盘法进行优化,获得晶形完美、衍射能力很强的hDHODH蛋白复合物单晶。用X射线衍射晶体,用CCP4、Coot软件解析结构,获得hDHODH蛋白复合物晶体结构。从解析的结构中可以看出,抑制剂与蛋白质的吻合度非常高,且抑制剂通过亲水的羧基端与蛋白质356位和147位的酪氨酸形成氢键网络。抑制剂的5元环与蛋白质359位的亮氨酸和360位的苏氨酸相互作用,使抑制剂与蛋白质牢固结合。该复合物晶体结构的顺利解析,将为开发新型特异性抗类风湿性关节炎药物提供重要基础。     

二氢乳清酸脱氢酶作为抗肿瘤靶点的研究进展

二氢乳清酸脱氢酶(dihydroorotate dehydrogenase,DHODH)是存在于线粒体内膜的一种含铁的黄素依赖酶,这种酶催化嘧啶核苷酸从头合成途径的第4步反应。而嘧啶核苷酸可用于DNA、RNA、糖蛋白和磷脂生物合成,对于细胞代谢和细胞增殖至关重要。近年来的研究表明,DHODH与多种肿瘤的发生、发展密切相关,抑制或下调DHODH可以降低肿瘤细胞增殖,诱导其凋亡或者增加其他靶点药物的抗肿瘤效果。该文结合所在实验室目前的研究成果及进展,就DHODH作为治疗恶性肿瘤靶点的相关机制及当前DHODH抑制剂的研究进展作一综述。     

八氢番茄红素脱氢酶的研究进展

类胡萝卜素是一类超过700种的萜烯基团类不饱和化合物的总称,根据结构可分为胡萝卜素族和叶黄素族,具有较高的营养价值。八氢番茄红素脱氢酶是类胡萝卜素生物合成途径中的首要限速酶,它参与催化无色的八氢番茄红素转变成有色类胡萝卜素,发挥着中心调控作用。不同生物源的八氢番茄红素脱氢酶在功能上呈现多样性,在大多数蓝细菌,藻类和高等植物的类胡萝卜素生物合成途径中,由Crt P,Crt Q和异构酶Crt H或PDS,ZDS和异构酶Z-ISO、Crt ISO共同参与番茄红素的形成,而在大多数微生物中只有Crt I-type一种酶来完成八氢番茄红素的脱氢反应,且根据脱氢步骤的不同分别可生成链孢红素、番茄红素或脱氢番茄红素。本文阐述了不同生物源八氢番茄红素脱氢酶的基因分离与鉴定,功能多样性及表达调控机制等最新研究进展,并进行了进化分析,为八氢番茄红素脱氢酶的深入研究及利用基因工程策略生产类胡萝卜素的应用提供重要信息。     

四氢嘧啶生物合成及其关键酶生化特性研究进展

相容性溶质是微生物分泌的一类高水溶性有机渗透物,以适应高盐度和高渗透压等极端环境。四氢嘧啶(ectoine)作为一种重要的相容性溶质,对核酸、蛋白、生物膜以及细胞具有修复和保护作用,广泛应用于化妆品、生物制剂、酶工业和医疗等领域,每公斤市场售价约为1 000美元,全球每年需求量高达1.5万t。嗜盐菌是四氢嘧啶天然合成的微生物来源,但其需在高盐培养基中生长,工业化生产存在设备腐蚀以及成本高昂等问题。随着功能基因组学、系统生物学和合成生物学的快速发展,利用代谢工程等手段构建四氢嘧啶高产细胞工厂成为当前重要的研究方向,工程化大肠杆菌的四氢嘧啶最高产量已达131.8 g/L,产率为1.37 g/(L‧h)。本文主要围绕四氢嘧啶的合成途径、关键酶的生化特性以及四氢嘧啶生物合成等方面进行综述,以期阐明其研究现状并为四氢嘧啶的工业生产提供思路和方向。     

四氢嘧啶羟化酶的研究进展

作为相容性物质,5-羟化四氢嘧啶不仅可以调节渗透压,还可以稳定蛋白结构,在医药、生物制造和化工行业具有广阔的发展前景。四氢嘧啶羟化酶属于Fe2+与2-酮戊二酸依赖型双加氧酶超家族,主要催化四氢嘧啶生成5-羟化四氢嘧啶。我们简要介绍了四氢嘧啶羟化酶的基因来源、活性检测、蛋白结构、催化机理及活性中心等方面的研究进展。     

二氢槲皮素的研究进展

二氢槲皮素是自然界中一种重要的黄酮类化合物,主要存在于高寒带落叶松的根部。由于其具有较好的抗氧化、抗肿瘤等生物学活性而被广泛应用于食品领域、工业领域和医药领域。然而,目前二氢槲皮素的工业化生产仍然依赖于传统的植物提取,原料稀缺、提取难度大、产率较低,阻碍了其工业化应用的推进。基于此,主要综述了二氢槲皮素的化学结构及性质、生物合成的分子机制、生物学活性以及生产工艺的研究进展,并对未来二氢槲皮素的相关研究趋势进行了展望,以期为日后二氢槲皮素的生物合成研究提供理论参考。     

相容性溶质四氢嘧啶及其羟基化衍生物的研究进展

四氢嘧啶类化合物是嗜盐以及耐盐菌胞内合成的一类能够抵御外界高盐胁迫的相容性溶质,概述了四氢嘧啶及其衍生物的理化特征以及在嗜盐微生物中抵御外界高渗透压的作用机理,主要阐述了四氢嘧啶类相容性溶质的生物合成途径、膜运输机理、分泌释放机制、高密度发酵生产等方面在细胞、分子水平上的最新研究进展以及前景展望。并且综述了四氢嘧啶类在精细化工、生物医药及生物制造等行业的应用研究以及发展前景,探讨了未来的研究方向。     

四氢嘧啶微生物合成与应用研究进展

极端环境微生物定义了生命的边界.为了适应各种极端环境,极端环境微生物通过合成许多独特的活性化合物来保护自己.四氢嘧啶就是其中一种代表性的保护性物质.它最早是从极端嗜盐菌中发现的,作为调节细胞渗透压的一类相容性溶质,可以帮助微生物适应高盐等恶劣环境.研究发现四氢嘧啶不仅是一种重要的渗透压调节剂,还是一种高效的生物保护剂,...     

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

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Pyrimidine biosynthetic enzymes in Babesia hylomysci

Gero A. M. and Coombs G. H. 1982. Pyrimidine biosynthetic enzymes in Babesia hylomysci. International Journal for Parasitology12: 377–382. The enzymes that catalyse the conversion of carbamylaspartate to UMP have been demonstrated in the rodent piroplasm, Babesia hylomysci and partially characterised. They were shown to be similar to the corresponding mammalian enzymes in that dihydroorotase, orotate phosphoribosytransferase and orotidine-5'-phosphate decarboxylase were soluble, whilst dihydroorotate dehyrogenase was membrane bound and appeared to be associated with a respiratory chain. Dihydroorotate dehydrogenase was found to have a K_m (l-DHO) of 21 μm and a pH optimum of 7.8. It was inhibited by analogs of ubiquinone and several pyrimidines, dihydroazaorotate being the most effective ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 17 μ}\text{m}$). It is concluded that Babesia parasites contain a functional de novo biosynthetic pathway for pyrimidines which provides a potential target at which to direct putative chemotherapeutic agents.     

Modifications induced by plasma of gestational hypertensive women on the Na+/K+-ATPase obtained from human placenta

Pyrimidines and purine (deoxy)nucleotides are the building blocks of DNA and RNA. Nucleoside diphosphate sugars, e.g. UDP-glucose, are the reactive intermediates in the synthesis of nearly all glycosidic bonds between sugars.In mammals the requirement for pyrimidines is met by UMP de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The exceptional compartmentation of the de novo synthesis with respect to mitochondrially-bound dihydroorotate dehydrogenase ('DHOdehase' or 'DHODH', EC 1.3.99.11) is one focus of the present work. DHODH activity was determined by the dihydroorotate-dependent oxygen consumption or by the UV absorption of the product orotate with mitochondria isolated from rodent and porcine tissues. For comparison, the cytochrome c and choline-dependent oxygen consumption of mitochondria from different tissues was measured. The highest specific activity of the rat DHODH was found in liver (2.3 × 10-3 µmol/min × mg protein) > kidney > heart. The application of known enzyme inhibitors Brequinar Sodium and Leflunomide for DHODH and sodium cyanide for cytochrome c oxidase verified the specificity of the activity tests used. The relation of DHODH activity versus that of cytochrome c oxidase revealed the lowest ratios in heart mitochondria and the highest in liver mitochondria. Since disorders in the mitochondrial energy metabolism could entail severe impairment of pyrimidine biosynthesis via respiratory-chain coupled DHODH, it is suggested to include improvement of pyrimidine nucleotide status in therapy protocols. (Mol Cell Biochem 174: 125–129, 1997)     

A small genetic region that controls dihydroorotate dehydrogenase in Drosophila melanogaster

A locus is described that controls levels of mitochondrial dihydroorotate dehydrogenase (EC 1.3.3.1) in Drosophila melanogaster. The effects of alleles of the locus, Dhod, are manifest in preparations from whole organisms as well as in partially purified mitochondrial preparations; however, other mitochondrial functions do not appear to be appreciably affected by Dhod genotypes. The locus maps near p in the proximal portion of the right arm of chromosome 3. Flies trisomic for a chromosome segment including that region display elevated enzyme levels, implying that an enzyme structural gene is in that vicinity. Furthermore, Dhod alleles are semidominant in heterozygotes, suggesting that the dosage-sensitive element detected in the trisomics is actually the Dhod locus. These findings are discussed relative to the role of dihydroorotate dehydrogenase in the de novo pyrimidine biosynthetic pathway and relative to other pathway mutants that have been described in Drosophila.This work was supported by NSF Grants PCM 76-17214 to W. Cohen and PCM 78-14164 To J. Rawls, as well as NIH Research Career Development Award 1 KO4 AM00676 to J. Rawls.