Functional and structural characterization of the microcompartments from mycobacteria and cyanobacteria

分枝杆菌和蓝藻微区室的功能和结构表征

• 批准号: RGPIN-2015-04045
• 负责人: Kimber, Matthew
• 金额: $ 2.77万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687121
• 关键词: Functional; structural; characterization; microcompartments; mycobacteria

Many bacteria can form bacterial microcompartments (BMCs), partitioning off large, specialized metabolic spaces using polyhedral protein shells. BMCs are built as large core of enzymes (90-400 nm in diameter) surrounded by a thin shell (2-6 nm). Carboxysomes, which encapsulate Rubisco and carbonic anhydrase to fix CO2 are the best characterized, but most examples mediate the catabolism of small metabolites that degrade via volatile, reactive aldehyde intermediates. Carboxysomes are of particular research interest as preliminary studies indicate that it may be possible to boost the performance of crops by engineering them into higher plants. Other potential uses for microcompartments include building cellular factories for making toxic compounds, or shielding therapeutic cargoes in the bloodstream prior to their delivery to target sites. Bioinformatics analyses distinguish at least 10 distinct BMC types, several of which are wholly uncharacterized. My lab seeks to understand the function, structure and large-scale organization of BMCs, working on two very different BMCs.******Our first target for study is the assembly of the enzymatic core of the ß-carboxysome, focusing on the key scaffold protein, CcmM. The small C-terminal domains of CcmM are known to interact with Rubisco, presumably by replacing one or more of the Rubisco small subdomains. We have expressed the RbcL/S/CcmM complex in E. coli and will characterize it structurally and kinetically. We will also try and understand the nucleation of the carboxysomal core by studying the interactions between longer CcmM constructs and Rubisco. The roles of the linkers between CcmM domains, and the possibility that Rubisco-Rubico interactions stabilize the core once CcmM has initiated its assembly are of particular interest. In parallel, we will study the complex formed between the N-terminal domain of CcmM with the essential structural protein CcmN, and with the ß-carbonic anhydrase, CcaA.******Our second target is a BMC of unknown function common in non-pathogenic Mycobacteria. Bioinformatics analysis indicates that the likely role of this BMC is degrading a small amine. We will determine the structures of all 9 proteins associated with this BMC, and characterize its overall function by searching for metabolites that induce its production. We will study the functions, structures and kinetics of the four encapsulated enzymes (an aminotransferase, an alcohol dehydrogenase, an aldehyde dehydrogenase, and a homoserine kinase-like protein), and work to understand the network of protein-protein interactions that efficiently recruit the enzymes to the BMC. We will also study the interactions between the shell proteins in order to understand this BMCs unusual shell architecture; there are far fewer shell proteins than most BMCs exhibit, with the only shell protein duplicated being one that is never duplicated in other shells.

许多细菌可以形成细菌微区室（BMC），使用多面体蛋白质壳划分大的专门代谢空间。BMC被构建为被薄壳（2-6 nm）包围的酶的大核心（直径90-400 nm）。羧化酶体，其封装Rubisco和碳酸酐酶以固定CO2是最好的特征，但大多数实例介导通过挥发性、反应性醛中间体降解的小代谢物的催化。羧化酶体具有特别的研究兴趣，因为初步研究表明，通过将其工程化到高等植物中来提高作物的性能是可能的。微隔室的其他潜在用途包括建造细胞工厂以制造有毒化合物，或在将治疗货物输送到目标部位之前将其屏蔽在血液中。生物信息学分析区分出至少10种不同的BMC类型，其中几种完全没有特征。我的实验室试图了解BMC的功能，结构和大规模组织，研究两种非常不同的BMC。我们研究的第一个目标是β-羧基体的酶核心的组装，重点是关键的支架蛋白CcmM。已知CcmM的小C-末端结构域与Rubisco相互作用，推测是通过替换一个或多个Rubisco小亚结构域。我们在大肠杆菌中表达了RbcL/S/CcmM复合物。大肠杆菌，并将其结构和动力学特征。我们还将尝试通过研究较长的CcmM构建体和Rubisco之间的相互作用来理解carboxysomal核心的成核。CcmM结构域之间的接头的作用，以及Rubisco-Rubico相互作用一旦CcmM启动其组装就稳定核心的可能性是特别感兴趣的。与此同时，我们将研究CcmM的N-末端结构域与必需结构蛋白CcmN以及与β-碳酸酐酶CcaA之间形成的复合物。我们的第二个目标是在非致病性分枝杆菌中常见的未知功能的BMC。生物信息学分析表明，这种BMC的可能作用是降解一种小胺。我们将确定与这种BMC相关的所有9种蛋白质的结构，并通过寻找诱导其产生的代谢物来表征其整体功能。我们将研究的功能，结构和动力学的四个封装酶（氨基转移酶，乙醇脱氢酶，乙醛脱氢酶，高丝氨酸激酶样蛋白），并努力了解蛋白质-蛋白质相互作用的网络，有效地招募酶的BMC。我们还将研究壳蛋白之间的相互作用，以了解这种BMC不寻常的壳结构;壳蛋白比大多数BMC表现出的要少得多，唯一复制的壳蛋白是在其他壳中从未复制的蛋白。