病原体Clostridum difficile的乙酰辅酶A合成通路中某些关键金属蛋白的基因克隆、重组表达与纯化及其结构与功能研究

【作者】 朱小飞；

【导师】 谭相石；

【作者基本信息】 复旦大学 ， 无机化学， 2011， 博士

【摘要】 厌氧病原体艰难梭菌(Clostridium difficile)能够直接感染人类导致结肠炎和结肠癌(简称为CDI),在北美和欧洲发现较多,发病率和死亡率较严重,目前在中国也已有报道。由于该病原体极度厌氧,对研究和疾病防治带来较大困难。艰难梭菌基因组测序结果表明：该病原体基因组中存在乙酰辅酶A合成通路(Wood-Ljungdah通路)中所有相关同源蛋白,主要包括甲基转移酶、钴铁硫蛋白、乙酰辅酶A合成酶和一氧化碳脱氢酶等。该通路是许多厌氧微生物中重要的能源和物质代谢通路之一,并且不存在于人体中。因此,这些蛋白(酶)有可能成为治疗Clostridium difficile所引发疾病的潜在药物靶点。本文研究的主要目的是为病原体中该通路的后续详细研究奠定基础,并为相关药物的开发提供设计思路。本文研究了病原体C. difficile 630的乙酰辅酶A通路中的四种关键酶(蛋白),包括甲基转移酶(MeTrCd,268个氨基酸)、钴铁硫蛋白(CoFeSPCd,包括两个亚基delta和gamma,分别含314个氨基酸和455个氨基酸)、乙酰辅酶A合成酶(ACSCd,708个氨基酸)和一氧化碳脱氢酶(CODHCd,639个氨基酸)。首先,我们研究了病原体C. difficile 630中编号为CD0727的甲基转移酶基因(MeTrCd),包括基因克隆、重组表达与纯化,成功建立了高效大肠杆菌表达纯化体系(蛋白产量可达50 mg每升TB培养基)；使用圆二色光谱对其二级结构进行了表征；通过序列和结构分析及生物信息学,得到了其同源模拟结构。研究了病原体C. difficile 630中编号为CD0725和CD0726(分别编码钴铁硫蛋白gamma和delta亚基)的基因,通过基因克隆、重组表达与纯化研究,成功建立了一套较为高效的重组表达体系；对CoFeSPCd的金属中心Fe4S4和钻胺素的化学重组进行了研究,并运用紫外可见光谱对其进行表征,实验结果表明我们通过重组得到的CoFeSPCd是具有完整金属中心的全蛋白。通过色氨酸荧光淬灭光谱测定了甲基四氢叶酸的底物结合常数,及其随pH值减小的构象变化现象。实验结果表明MeTrCd的底物结合能力与同源蛋白MeTrMt(来源于嗜热菌Moorella thermoaceticum的MeTr)相比有显著下降。该差异可能是由MeTrCd中底物结合位点腔His1l的存在造成的。色氨酸荧光淬灭光谱实验表明MeTrCd存在pH依赖性的构象变化性质。该变化导致蛋白疏水区域的色氨酸残基溶剂暴露程度加大,从而有利于底物结合及完成催化功能。通过厌氧条件下的截流光谱技术详细研究了MeTrCd的甲基转移反应动力学,得到了MeTrCd与外源性CoFeSPMt(来源于嗜热菌Moorella thermoaceticum的CoFeSP)和钻铵素反应的米式方程动力学参数。通过厌氧条件下的截流光谱技术检测了CoFeSPCd接受甲基的功能,并通过改变CoFeSPCd的浓度得到了以CoFeSPCd为底物的MeTrCd催化甲基动力学常数。研究发现分别使用CoFeSPMt、钴铵素和CoFeSPCd作为催化底物,无论是催化速率常数(Kcat)还是催化效率(Kcat/Km)都遵循这样一个规律：CoFeSPMt<CoFeSPCd<钴胺素。该规律进一步验证了我们的推论即当使用MeTrCd-CoFeSPCd同源反应体系时,其相互作用的增强有利于甲基转移反应的完成。采用计算机模拟分子对接研究对MeTr和CoFeSP的相互作用进行了研究,结果表明MeTr和CoFeSP相互作用有可能采取侧面进攻(Conform3)的方式完成,并且CoFeSPCd与MeTrCd相互作用时的交界面处氨基酸的盐桥、氢键等起了重要的稳定作用。本文详细研究了乙酰辅酶A合成酶。首次建立了病原体C. difficile 630乙酰辅酶A合成酶(ACSCd)的大肠杆菌体外重组表达纯化体系；通过金属含量分析、紫外可见光谱以及EXAFS等光谱研究表明ACSCd在结构上具有ACSMt(来源于嗜热菌Moorella thermoaceticum的ACS)的典型金属活性中心A-cluster;活性测定实验表明ACSCd的乙酰辅酶A合成活性速率常数为0.04μM min-1mg-1、甲基转移酶活性速率常数为0.26±0.05 min-1；对ACSCd的抑制剂进行了初步的研究,并发现三种抑制剂均能有效抑制其活性；离体抑菌活性研究进一步验证了它们对病原体C.difficile的抑制效果。这一研究具有十分重大的理论意义和应用价值,有可能成为寻找治疗CDI疾病药物的一个新策略。此外,我们对一氧化碳脱氢酶的基因克隆、蛋白质重组表达纯化进行了初步的研究探索。一氧化碳脱氢酶(CODH)的功能是催化CO和CO2的可逆氧化还原,它是乙酰辅酶A通路中的重要金属酶之一,但是该蛋白很容易以包涵体沉淀的形式存在,人们一直未能建立该蛋白的大肠杆菌表达体系。本文通过构建多个原核表达质粒,对嗜热菌Moorella thermoacetica的CODHMt以及病原体CODHCd的重组表达纯化进行了一系列的探索,我们得到了含有麦芽糖融合标签的蛋白。这一研究为将来详细研究CODH的结构与功能奠定了基础。总之,本文对艰难梭菌(Clostridium difficile)乙酰辅酶A合成通路中的四种关键酶(蛋白)进行了基因构建、重组表达与纯化的研究探索,成功得到了其中3种酶(蛋白)的高效表达纯化体系,并进行了详细的结构、功能与性质及抑制剂的研究。该研究不仅增进了人们对该病原体的认识和对乙酰辅酶A合成通路的了解、而且为开发新型治疗CDI药物提供了理论依据和思路。更多还原

【Abstract】 Clostridium difficile is a ubiquitous anaerobic and spore-forming pathogen. It can produce toxins A and B, and then causes acute illnesses called Clostridium difficile infection (CDI) in humans, ranging from severe diarrhea, antibiotic-associated colitis, pseudomembranous colitis, toxic megacolon, intestinal perforations, and even death. It was found that the genomic DNA of the pathogen contains several genes encoding proteins involved in acetyl-coenzyme A synthesis of Wood-Ljungdahl pathway. The pathway for catabolism and anabolism of the pathogen involves a series of critical enzymes such as formate dehydrogenase, corrinoid-dependent methyltransferase, corrinoid iron-sulfur protein, and acetyl-CoA synthase. Each of the four metalloenzymes plays a critical role in the Wood-Ljungdahl pathway. Thus, these metalloenzymes could be a selective and promising target for the development of new antibiotics agents. Compounds targeting this enzyme should avoid multiple resistance with currently used antibiotics agents. It could be a new strategy to find special antibiotics for the treatment of CDI.In this study, we investigated some key metalloenzymes including methyltransferase (MeTrCd,268 aa), Corrinoid iron-sulfur proteins (CoFeSPCd, two subunits included:delta,314 aa and gamma,455 aa), acetyl-coenzyme A synthase (ACSCd,708 aa) and carbon monoxide dehydrogenases (CODHCd,639 aa).Corrinoid-dependent methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron-sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from Clostridium difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50 mg MeTr was produced per liter TB medium. The recombinant methyltransferase was well characterized by SDS-PAGE, gel filtration chromatograpgy, enzyme assay and far-UV circular dichroism (CD). Furthermore, steady-state kinetics were studied using exogenous cobalamin and CoFeSP from Moorella thermoacetica (CoFeSPMt) as a substrate by stopped-flow method. MeTrCd showed pH dependent conformational change and methyl transfer activity. The kinetic studies of MeTrCd were fitted to the Michaelis-Menten equation, yielding values of kcat(77.6 s-1) and kcat/Km (1.33μM-1s-1) for hydroxycobalamin, and kcat(2.63 s-1) and kcat/Km(0.15μM-1s-1) for CoFeSPMt.CoFeSP acts as a transformer in the Wood-Ljungdahl pathway, which belongs to B12-dependent enzymes. In this study, the putative gamma and delta gene of CoFeSPcd from clostridium difficile was firstly expressed in E. coli. High yield of recombinant gammaCd and deltaCd was achieved in this expression system, which was purified efficiently by affinity chromatography. Furthermore, we got the holo-protein using the reconstitution of Fe4S4 and cobalamin. Then, steady-state kinetic was studied using CoFeSPCd as a methyl accepter by stopped-flow method, yielding values of kcat(43.7 s-1) and kcat/Km (0.51μM-s-1). The results of kinetic parameters, using hydroxycobalamin, CoFeSPMt and CoFeSPCd as a substrate respectively, were suggested that the interaction between CoFeSP and MeTr was a key step in the methyl transfer reaction.Acetyl-CoA synthases catalyses the condensation of three substrates:CoA, CO, and a methyl group from the methylated corrinoid iron-sulfur protein (CH3-CoFeSP), to produce acetyl-CoA. In this study, the target gene was subcloned into the vector pET30a (Novagen) to generate a C-terminal His-tagged protein. Based this, we established a recombinant expression system for ACSCd in E. coli that enabled a one-step purification of active enzyme under anaerobic conditions. The A-cluster, [Fe4S4][NipNid], existed in ACSCd was confirmed by structural modeling, metal analysis, and UV-Vis spectra of the characteristic feature of [Fe4S4] cubane. The Nip, as a labile metal, can be removed by treatment with chelators but this treatment results in the loss of ACS activity. Three bidentate chelators (10-phenanthroline, 8-hydroxyquinoline, and 2,2-dipyridyl) showed inhibition effects on ACSCd methyl group transfer and acetyl-coenzyme A synthesis activity. These inhibitory results were confirmed by further antibacterial activity assay against Clostridium difficile, though 8-hydroxyquinoline has a weak effect. These results played a solid base for the future research of high throughput screening to find valuable inhibitors, and it might be a new strategy to find new antibiotics for the treatment of CDI.At last, we tried to establish a recombinant expression and purifaction system for CODH. CODH catalyzes the reversible oxidation and reduction between carbon monoxide and carbon dioxide. CODHs from Carboxydothermus hydrogenoformans, Rhodospirillum rubrum and Moorella thermoacetica have been well studied. X-ray crystal structures of all three enzymes (CODHRr, CODHCh and CODHMt) were determined between 2001 and 2007. However, the reaction mechanism is probably the most intriguing. We constructed several plasmids and tried to establish a highly effective expression and purification system for CODHmt and CODHCd.Luckily, we got the MBP-fusion protein. All these studies will provide useful information for future research.In summary, we described for the first time several means of expressing at high level and purifying methyltetrahydrofolate- and corrinoid-dependent methyl-transferase, corrinoid iron-sulfur protein and acetyl-coenzyme A synthase from human pathogen C. difficile. Their structures and functions were investigated by spectroscopy, enzyme kinetics, and homology structure modeling. Inhibitors based on the enzyme ACSCd assay showed effective antibacterial activity for the pathogen C. difficile. In particular, this study provides a solid basis for the studies of potential drug target like ACSCd, which may apply to drug discovery for the treatment of CDI.更多还原