Functional and structural characterization of the microcompartments from mycobacteria and cyanobacteria

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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.