【作者】 王飞飞；

【导师】 张雁；

【作者基本信息】 天津大学 ， 药学， 2020， 硕士

【摘要】 嘧啶是一种含氮杂环化合物,是组成核酸和脱氧核酸单体核苷酸和脱氧核糖核苷酸的成分,因此嘧啶是生命的必须成分,广泛的存在于自然界中,微生物利用降解嘧啶来获得碳和氮以及能量。现已被证实的微生物中尿嘧啶代谢途径有三种,分别是尿嘧啶降解的还原途径,Rut途径和氧化途径。其中,研究最广泛的是尿嘧啶的还原代谢途径,在该途径中Pyd A(二氢嘧啶脱氢酶)负责催化细菌和真核生物中还原性嘧啶降解(Pyd)途径的第一步,把尿嘧啶还原为二氢尿嘧啶。Pyd A是一种含有铁硫簇的黄素酶,目前文献报道的Pyd A同源物催化尿嘧啶还原为二氢尿嘧啶的反应其电子来源是NAD(P)H。Pyd A含有两个黄素辅基FAD(黄素腺嘌呤二核苷酸)和FMN(黄素单核苷酸),尿嘧啶还原发生在FMN的位点,而NAD(P)H氧化发生在FAD的位点,两个铁氧还蛋白域介导了位点间的电子转移。本课题组在研究嘧啶降解途径相关酶时发现,与已报道的Pyd A结构相比,还原铬梭菌(Clostridium chromiireducens)中的Pyd A(Pyd Ac)缺少FAD结构域,这表明它无法利用NAD(P)H催化尿嘧啶的还原反应。因此我们作出假设:Pyd Ac能够使用铁氧还蛋白(Fdx)作为电子源催化尿嘧啶还原。在这里,我们报道C.chromiireducen中重组蛋白Pyd Ac的生化特征。本文通过生物信息学分析比较了Pyd Ac与已报道的S.scrofa和短杆菌的Pyd A同系物的结构,结果表明Pyd Ac包含一个FMN结构域和两个Fdx结构域,但缺少FAD和NADPH域。接下来我进行了一系列生化实验,首先克隆表达纯化Pyd Ac以及体外重构建铁硫簇然后进行酶活性检测,通过显色反应和高效液相色谱-质谱联用(LC-MS)技术检测产物二氢尿嘧啶的生成确认了Pyd Ac的催化活性;Pyd Ac可以利用甲基紫精以及Fdx获得还原力催化尿嘧啶的还原降解;C.chromiireducens在以尿嘧啶为唯一氮源的特定培养基中生长结果,说明了尿嘧啶可以作为唯一的氮源支持C.chromiireducens的能量代谢。通过上述实验结果可以得出结论:在C.chromiireducens中,Pyd Ac可以从Fdx获得电子,催化尿嘧啶的还原代谢。此外,通过生物信息学分析发现了Pyd Ac的变型部分存在于厌氧梭状芽孢杆菌的细菌中,其中包括C.chromiireducens,这些菌的生理代谢依赖于Fdx进行电子传递。因此我们本课题的发现与这些菌的生理特性吻合,该发现对研究微生物代谢及合成生物学的多样性具有重要理论意义,并具有潜在的应用价值。更多还原

【Abstract】 Pyrimidines are nitrogen-containing heterocyclic compound that are components of ribonucleic acid and deoxyribonucleic acid,and their precursor nucleotides and deoxyribonucleotides.Therefore,pyrimidines are essential to life,and are widespread in nature,and microorganisms use pyrimidine degradation to obtain carbon and nitrogen and energy.Three pyrimidine degradation pathways in bacteria have been discovered: the reductive pathway,Rut pathway and oxidative pathway.Among them,the most extensively studied is the reductive metabolic pathway.Dihydropyrimidine dehydrogenase(Pyd A)catalyzes the first step of the reductive pyrimidine degradation(Pyd)pathway in bacteria and eukaryotes.Pyd A homologs studied to date catalyze the reduction of uracil to dihydrouracil,coupled to the oxidation of NAD(P)H.Uracil reduction occurs at a flavin mononucleotide(FMN)site,and NAD(P)H oxidation occurs at a flavin adenine dinucleotide(FAD)site,with two ferredoxin(Fdx)domains thought to mediate inter-site electron transfer.In the investigation of Pyd pathway,we noticed that a Clostridial Pyd A homolog(Pyd Ac)lacked the FAD domain,and is present in a Pyd gene cluster in the strict anaerobic bacterium Clostridium chromiireducens.We therefore hypothesized that Pyd Ac is able to catalyze uracil reduction using reduced Fdx as the electron source.Here we report the biochemical characterization of recombinant Pyd Ac.To investigate this hypothesis,we first compared the domain structure of Pyd Ac with that of the previously studied Pyd A homologs from S.scrofa and Brevibacillus agri through bioinformatics analysis.The result showed that it contains the FMN domain and two Fdx domains in a single ORF,but lacked the FAD and NADPH domains.A variety of experiments were also performed.C.chromiireducens Pyd Ac was recombinantly produced in E.coli,and purified to near homogeneity,then followed reconstitution of purified Pyd Ac in vitro.Colorimetric activity assays combined with high-performance liquid chromatography-mass spectrometry(LC-MS)were carried out to detect the production of dihydrouracil and thereby confirmed the activity of Pyd Ac.Pyd Ac can rely on either methyl viologen or Fdx to obtain reducing power to catalyze uracil reduction.C.chromiireducens could grow in defined medium that clarified uracil could be used as the sole nitrogen source for bacterial growth.In addition,it was discovered through bioinformatics that the majority of Pyd Ac sequences belong to strict anaerobic bacteria in the order Clostridiales,including C.chromiireducens Pyd Ac.These homologs likely do not use NAD(P)H as a reductant and may use reduced Fdx as a reductant,consistent with the physiological characteristics of these bacteria.Moreover,the findings not only have vital theoretical significance for studying the diversity of microbial metabolism and synthetic biology but have potential application value.更多还原