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纤维小体组成模块的发掘及其结构功能研究
陈超
导师冯银刚
2015-11
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化学与分子生物化学
关键词纤维小体 对接模块 膨胀因子 Anti-sigma 核磁共振 热纤梭菌
摘要

纤维小体是通过对接模块-粘连模块的相互作用将纤维素酶、半纤维素酶等催化组分组装在一起的多酶复合体,是自然界中降解木质纤维素资源最高效的体系之一。纤维小体整体的结构组成已经基本清楚,但由于基因组中许多特殊纤维小体组分的陆续出现和之前研究存在的矛盾之处等原因,纤维小体的组成模块仍有许多未知和模糊的地方,对纤维小体组成模块的继续发掘不仅可以帮助我们了解其高效的底物降解机制,还可以为其生产菌株的改造提供线索。因此,本论文从纤维小体组装、特殊催化区域及其表达调控三个方面着手,对4种纤维小体相关模块或结构域进行了以下研究。 (1) 热纤梭菌对接模块的核磁溶液结构的研究 纤维小体酶上的对接模块可以与脚架蛋白中的粘连模块紧密结合,从而在纤维小体的组装中发挥重要作用。之前解析的热纤梭菌Cel48S的I型对接模块的核磁溶液结构只利用了二维1H-1H NOESY和三维的1H-15N-1H NOESY实验所得的距离约束,与后来的晶体复合物中的I型对接模块的结构有较大的差异,因此,对接模块结合粘连模块后是否发生构象的变化便成为了一个待解决的问题。我们利用1H -15N-1H和1H- 13C-1H NOESY实验重新对该对接模块的核磁结构进行了解析。新解析的结构和晶体复合物中对接模块的结构相似,表明对接模块是一种固有的依靠钙离子的结构,它不会因粘连模块的结合诱导而发生构象变化。 通过序列分析,我们还在热纤梭菌中还发现了特殊的、保守的、由两个I型对接模块串联而成的双对接模块,可能使我们对纤维小体的组成和组装方式产生新的认识。初步的功能研究表明其确实具有不同的粘连模块结合特性。当把它拆分成两个独立的对接模块时,便丧失了大部分的粘连模块结合能力,表明其完整性对于其功能至关重要。此外,我们收集了双对接模块用于结构解析的核磁数据,目前已经完成了大部分的化学位移指认并获得了它的二级结构信息。 (2) 纤维小体类膨胀因子模块功能的鉴定 通过基因组分析发现,纤维小体生产菌株Clostridium clariflavum DSM 19732中含有两个类膨胀因子蛋白,并且它们都含有对接模块,表明它们同时还是纤维小体的组成部分。通过对这两个蛋白的类膨胀因子模块的研究发现,它们和其它细菌膨胀因子一样,本身不具备纤维素的水解活性,但是它们可以促进游离的天然纤维小体降解纤维素产糖,证明了它们膨胀因子的功能。当它们被组装到人工纤维小体以后,这种促进产糖的协同作用则会进一步的提升。此外,序列和结构分析表明,它们的作用机制与其它细菌膨胀因子相同。这两个纤维小体类膨胀因子模块的发掘为纤维小体底物转化效率的提高提供了新的方式。 (3) Anti-sigma因子的两个结构域结构和功能的初步研究 Sigma-anti-sigma因子是热纤梭菌纤维小体最主要的表达调控因子,anti-sigma因子通过感知底物的变化将信号传递到细胞内从而影响纤维小体相关基因的转录,但是其信号传递方式并没有研究清楚。我们对热纤梭菌一个典型anti-sigma因子的胞内N端区域及周质空间区域进行了初步的研究,获得了这两个结构域的最终核磁结构,为anti-sigma因子的研究奠定了结构基础。我们还利用遗传操作技术获得了周质空间区域缺失的突变株,该区域的缺失可以导致一些纤维小体基因的转录水平上调,可能与其缺失破坏了anti-sigma因子的功能或细胞内定位有关。 本论文对纤维小体相关模块: I型对接模块、双对接模块、类膨胀因子模块和anti-sigma因子的胞内N端区域及周质空间区域进行了发掘,研究并阐述了它们的结构和(或)功能,为纤维小体的结构生物学研究、纤维小体高效的底物降解机制及纤维小体生产菌株的遗传改造奠定了基础。

其他摘要

Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions. Cellulosome is one of the most efficient systems for the degradation of lignocellulosic resources in nature. The whole structure of the cellulosome has been well known, however, many special components have appeared successively in the genomic sequencing, and there are contradictions in the previous studies and so on, the functions and structures of several cellulosomal components are kept unknown or uncertain, which requires continuous excavation. The study on cellulosomal modules can not only help us understand its efficient mechanism for substrate degradation, but also provide the basis and clues for the modification of the cellulosome-producing strains. This thesis, therefore, focused on the following three aspects: the assembly of cellulosome, the catalytic regions, and the expression regulation of the cellulosome, and four different cellulosomal modules were chosen as the main research objects, as follows. (1) Study of the NMR solution structures of the dockerin modules from Clostridium thermocellum Dockerin modules of the cellulosomal enzyme subunits play an important role in the assembly of the cellulosome by binding tenaciously to cohesin modules of the scaffoldin subunit. A previously reported NMR-derived solution structure of the type-I dockerin module from Cel48S of Clostridium thermocellum, which utilized two-dimensional homonuclear 1H-1H NOESY and three-dimensional 15N-edited NOESY distance restraints, displayed substantial conformational differences from subsequent structures of dockerin modules in complex with their cognate cohesin modules, raising the question whether the source of the observed differences resulted from cohesin-induced structural rearrangements. We determined the solution structure of this dockerin based on 15N- and 13C-edited NOESY-derived distance restraints again. The structure adopted a fold similar to X-ray crystal structures of dockerin modules in complex with their cohesin partners, indicating that it does not undergo appreciable cohesin-induced structural alterations but rather assumes an inherent calcium-dependent cohesin-primed conformation. We then found a special and conserved double-dockerin module consisting of two tandem type-I dockerins in C. thermocellum through sequence analysis, which maight provide us new insights into the composition and assembly of cellulosome. Preliminary functional study revealed that it indeed possesses different binding properties with different cohesins. When it was splited into two independent dockerin modules, it lost most of the cohesion binding capacities, indicating that the integrity is essential for its function. Furthemore, NMR data of the double-dockerin module was collected and would be used for the calculation of its structure, most of the chemical shifts of it have been assigned and its secondary structure has also been got. (2) Functional identification of the cellulosomal expansin-like modules. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins contain a dockerin module, which suggests that they are components of the cellulosome. Through the study of the expansion-like modules of these two proteins, they were found to have no hydrolytic activities like other bacterial expansin-like proteins, but could facilitate the degradation of cellulose via synergistic effects with free native cellulosomes, indicating the cellulosomal expansin-like proteins have the function of expansin. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. The excavation of the function of these two expansin-like modules in the cellulosome may provide a new way to improve the efficiency of the substrate conversion of cellulosome. (3) Preliminary study of the structure and function of two domains in anti-sigma Sigma-anti-sigma is the most important regulatory factor for the expression of cellulosomal genes of C. thermocellum, in which the anti-sigma factor could sense the change of substrate and then transmit the regulation signal to the internal of cell and interact with sigma fator to affect the transcription of the related cellulosomal genes. However, the signal transduction mechanism is unclear. The intracellular N-terminal domain and periplasmic domain of a typical anti-sigma factor of C. thermocellum were analyzed, and finally the refined NMR stuctures of them were obtained, which may provide the structrual foundation for the further study of anti-sigma factors. The periplasmic domain was disrupted using a previously developed genetic manipulation technology, and the transcription of some cellulosomal genes of the obtained mutant was analyzed. General up-regulation of the selected genes in this mutant was observed, which indicated the the lack of periplasmic domain might cause the inactivation of the anti-sigma or the disruption of its cellular localization. In this thesis, some excavations were carried on these cellulosome related modules: type-I dockerin module, double-douckerin module, expansin-like modules and the intracellular N-terminal domain and periplasmic domain of a anti-sigma factor. The structures and (or) functions of them were studied, which provide the foundation of the structural biology of the cellulosome, the efficient mechanism for substrate degradation of the cellulosome and the modification of the cellulosome-producing strains.

作者部门代谢物组学团队
学科领域生物
公开日期2020-12-01
学位类型博士 ; 学位论文
语种中文
文献类型学位论文
条目标识符http://ir.qibebt.ac.cn/handle/337004/8072
专题代谢物组学研究组
作者单位中国科学院青岛生物能源与过程研究所
推荐引用方式
GB/T 7714
陈超. 纤维小体组成模块的发掘及其结构功能研究[D]. 北京. 中国科学院研究生院,2015.
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