新型糖苷水解酶的结构和功能研究

QIBEBT-IR > 代谢物组学研究组

	新型糖苷水解酶的结构和功能研究
	东升
	2016-07
关键词	糖苷水解酶 Β-葡萄糖苷酶地衣聚糖酶进行性纤维素内切酶晶体结构结构与功能
学科分类	生物学
报告类别	专题报告
中文摘要	The global energy crisis and dioxide emission have demanded development of renewable energy. Lignocellulose is an important renewable resource, however it is hard to be used at a low cost due to its inherent anti-degration barrier. Glycoside hydrolases, which can hydrolyze the glycoside bond, have important applications in the utilization of lignocellulose. Glycoside hydrolyases are becoming more and more important according to the developments of carbohydrates biology. Protein crystallography is used to solve the structures of 3 novel glycoside hydrolases, and structure and function studies were done furtherly to elucidate the catalytic mechanisms of these enzymes in this dissertation. (1) structure and function of a glucoside hydrolase from Nannochloropsis Nannochloropsis spp. are emerging as a model system for oleaginous microalga on research and industrial production. Previous studies have shown that cabarhydrate could be converted to triacylglycerol (TAG) as storage material. Glycosidases play important roles during the transformation of carbohydrates. In this dissertation, glycosidase g6403 was taken as research object, whose transcriptional level was among the highest ones. Bioinformatics studies showed that this β-glucosidase was belonged to GH1 family and located at mitochondria. The crystal structure of this enzyme revealed that it had a typical (β/α)8 structure. There were some sructural differences between g6403 and homologous proteins at the substrate binding pocket which was an adaption of the substrate specificity. Characterization of g6403 showed that this enzyme adapted the marine environments well. (2) structure of lichenase F32EG5 with ligand F32EG5 was the first reported lichenase that belonged to GH5. The crystal structure of inactivated mutant F32EG5 (E188Q) with a ligand cellotetraose (G4) was solved to elucidate the catalytic mechanism of this enzyme. The substrate G4 binding in the catalytic cleft of F32EG5 fron positon -3 to +1. The recognition of substrate at -1 and -2 was quit strictly while relatively weak at subsite -3 and +1. The substrate adopts a skew-boat conformation at subsite -1 and the extend direction of the glycan change with a large angle. This might be the reason of the substrate preference of F32EG5. (3) structure of a processive endo-cellulase CHU2103 from Cytophaga hutchinsonii Cytophaga hutchinsonii can efficiently degrade cellulose by an unknown strategy, which is quite different from the two well-studied strategies of cellulose degradation for cellulolytic microorganisms. The aerobic fungi degrade cellulose by secreting a series of free cellulases, while most anaerobic cellulolytic microorganisms produce large multienzyme complexes, cellulosomes, bound to the outer surface of the organisms. Furthuemore, most of the cellulases in C. hutchinsonii do not contain any carbohydrate-binding modules (CBMs). The structure of CHU2103, which is a processive endo-cellulase from C. hutchinsonii, was solved in this study. The CHU2103 has a shallow substrate binding cleft while a deep active center pocket which is comparatively uncommon for endo-cellulases. CHU2103 maight also be an exoglucanases due to its special structure which lead to more soluable reducing sugars than insoluable reducing sugars. The crystal structures of processisive endo-cellulase CHU2103, lichenase F32EG5 with a ligand G4 and β-glucosidases g6403 are solved in this dissertation and their structural basis for function are determined. This work will promote the further study of these enzymes and provide theoretical basis for the functional renovations. Our lab have purified CohA and DocA through Escherichia coli, studied their resolution structure through nuclear magnetic resonance (NMR), and tested their interaction through surface plasmon resonance (SPR), too. My work in this thesis dose further study about assembly CohA with DocA, and enzyme activity of assembled complexes. We select one cohesin(CohC) and one dockerin(DocC) from Clostridium cellulolyticum as control.
英文摘要	发展可再生能源是当前人类社会缓解能源危机，减少温室气体排放的重要应对策略之一。木质纤维素是一种重要的可再生资源，但是其存在一些固有的抗降解屏障，使其低成本的大规模应用目前还很难实现。木质纤维素等多糖类生物质资源的利用离不开糖苷水解酶的作用。糖苷水解酶是一类可以降解糖苷键的酶类,随着糖类生物学地位的日渐突出，糖苷水解酶的发现与研究也越发重要。为了阐明新型的糖苷水解酶的作用机理，本论文利用蛋白质晶体学的相关技术，对它们进行结构功能的研究。本实验室的前期工作已经通过核磁共振方法解析了CohA和DocA的单独溶液结构，通过表面等离子共振技术对CohA-DocA之间的相互作用强度进行了测定。本论文在此基础上进一步研究了CohA-DocA之间的相互作用对纤维小体组装与组装后功能的影响。以解纤维梭菌的粘连模块（CohC）和对接模块（DocC）作为对照组。本论文的工作主要包括对所需组件的基因克隆与蛋白表达纯化，实验分析相互作用对组装的影响以及对组装后复合物协同效应的影响。（1）微拟球藻胞内糖苷水解酶的结构功能研究微拟球藻是一种重要的产油微藻，作为微藻产油研究的模式藻株，研究者已经对其开展了大量的研究。此前的研究报道认为，微拟球藻胞内的物质存在转化通路，特别是多糖类物质可以转化为油脂储存。该过程中糖苷水解酶起着至关重要的作用。本论文选择了微拟球藻胞内转录水平最高的一种糖苷水解酶g6403进行了相关研究。生物信息学分析表明该酶是GH1家族的β-葡萄糖苷酶，该酶是定位于微拟球藻线粒体上的，与其物质能量代谢密切相关。通过蛋白质晶体学技术解析了g6403的蛋白结构，它有典型的糖苷水解酶的(β/α)8结构。其底物结合口袋与之前研究过的酶有一定的差异，可能与其独特的底物降解偏好性相适应。进一步对其酶学性质的研究表明其最适的酶活作用条件是与其细胞定位高度适应的。（2）地衣聚糖酶F32EG5与底物复合物的结构研究 F32EG5是首次报道的GH5家族的地衣聚糖酶。为了揭示其独特的底物识别和催化机理，本研究论文解析了其无活性突变体E188Q和纤维四糖底物G4的复合物的晶体结构。结果表明，底物G4结合在酶的-3到+1位。F32EG5在-1和-2位对G4底物有严格的识别作用，在-3位和+1位的结合比较弱。在-1位的糖环呈现出扭曲的船式构象，糖链发生了较大角度的扭曲，这可能是其对β-1，3降解偏好性的重要原因之一。（3）哈氏噬纤维菌纤维素内切酶CHU2103的结构研究哈氏噬纤维菌可以实现对纤维素的高效降解，但是其纤维素降解机制既不同于厌氧菌的纤维小体机制，也不同于真菌的游离酶系，并且其编码的纤维素酶缺乏CBM结构域，这都预示着其有独特的纤维素降解机制。CHU2103是哈氏菌编码的一种进行性内切酶，本研究通过解析其晶体结构，发现它有一个很浅的底物结合裂隙，且其中的芳香族氨基酸的数目特别多，这可能是其缺乏的CBM的一种适应性机制。另外，我们发现其活性中心有一个较深的口袋结构，不同于常见的纤维素内切酶，我们推测其具有纤维素外切活性，导致其产物中可溶性还原糖明显多于不可溶性还原糖。本论文对进行性纤维素内切酶CHU2103，地衣聚糖酶F32EG5+G4，以及β-葡萄糖苷酶进行了结构解析，阐述了它们独特的生化性质的结构基础，为对其进行进一步的性质研究和功能改造提供了重要的理论依据和基础。
文献类型	研究报告
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9809
专题	代谢物组学研究组
推荐引用方式 GB/T 7714	东升. 新型糖苷水解酶的结构和功能研究. 2016.

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