Functional and structural characterization of the microcompartments from mycobacteria and cyanobacteria

许多细菌可以形成细菌微室(BMC)，使用多面体蛋白质壳来分隔大型的专门代谢空间。骨髓基质细胞是由直径90-400 nm的大酶核构成的，周围有一层薄的外壳(2-6 nm)。包裹Rubisco和碳酸酐酶以固定二氧化碳的羧基体是最有特点的，但大多数例子通过挥发性、反应性的醛中间体来调节小代谢物的分解代谢。羧基体是特别有研究意义的，因为初步研究表明，通过将它们改造成高等植物，可能会提高作物的表现。微隔室的其他潜在用途包括建造制造有毒化合物的细胞工厂，或者在将治疗物品运送到目标位置之前屏蔽血液中的治疗物品。生物信息学分析识别出至少10种不同的BMC类型，其中几种完全没有特征。我的实验室致力于了解BMC的功能、结构和大规模组织，致力于两个截然不同的BMC。 我们研究的第一个目标是组装ç-羧体的酶核心，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，推测是通过取代一个或多个Rubisco小亚域。我们已经在大肠杆菌中表达了rbcL/S/CcmM复合体，并对其进行了结构和动力学性质的研究。我们还将通过研究较长的CcmM结构和Rubisco之间的相互作用来尝试和理解羧体核心的成核。CcmM结构域之间的连接子的作用，以及一旦CcmM启动其组装，Rubisco-Rubito相互作用稳定核心的可能性是特别感兴趣的。同时，我们将研究CcmM的N-末端结构域与基本结构蛋白CcmN，以及与ç-碳酸酐酶CCAA之间形成的复合体。 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。我们将确定与该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。我们将研究四种被包裹的酶(转氨酶、乙醇脱氢酶、乙醛脱氢酶和高丝氨酸激酶样蛋白)的功能、结构和动力学，并努力了解有效地将这些酶招募到BMC的蛋白质-蛋白质相互作用网络。我们还将研究壳蛋白之间的相互作用，以了解这种不寻常的壳结构；比大多数骨髓细胞所展示的壳蛋白少得多，唯一复制的壳蛋白是一种从未在其他壳中复制的壳蛋白。

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.