| Abstract: | Our understanding of gene essentiality in Mycobacterium tuberculosis (Mtb) has improved over the years. The introduction of these gene products into target-based drug discovery campaigns depends, in part, on whether or not their biological functions are known. Many essential Mtb genes are yet to be assigned biological roles. Certain members of the aminotransferase family of pyridoxal phosphate (PLP) dependent enzymes are a good example. Aminotransferases are at the convergence of carbon and nitrogen metabolism. In chapters 1 and 2, we combined metabolomics, enzymology, and macromolecular crystallography to assign functions to two essential mycobacterial aminotransferases Rv3772c and Rv1178, respectively. We show Rv3722c is an L-aspartate aminotransferase (AspAT) with L-kynurenine side activity. Crystal structures of Rv3722c in complex with L-glutamate and L-kynurenine provided the basis for dual ligand recognition. We also show Rv1178 is a mycobacterial N-succinyl-L, L-diaminopimelate aminotransferase (DapATase) with a minor AspAT side activity. Crystal structures of Rv1178 ligand-free and bound to N-succinyl-L-2-amino-6-ketopimelate provided the structural determinants governing the recognition of this tricarboxylic acid substrate. The active site of Rv1178 has a narrow pocket extending beneath the plane of the pyridine ring of PLP. In the ligand-bound structure, this pocket binds the succinyl moiety of the substrate. Finally, in chapter 4, we employed a series of biochemical and structural studies to characterize Rv3208A, a conserved and hypothetical protein with a predicted secondary structure similar to that of the sRNA chaperone Hfq. Rv3208A has a novel ¹̐Ł1¹̐Ø5 tertiary structure that oligomerizes into either a homo-octameric or homo-hexadecameric toroid. A rv3208a deletion strain of Mtb MC2 7000 grows faster than wildtype in at least two carbon-defined growth media. The knockout strain is also resistant to the cell wall targeting drug polymyxin B. Taken together, the work in chapters 2 and 3 updates our current view of nitrogen metabolism in Mtb. Assigning functions to Rv3722c and Rv1178 also paves the way for these enzymes to be considered candidates for target-based drug discovery campaigns. Lastly, the work in chapter 4 implicates Rv3208A in growth regulation in mycobacteria and provides impetus for future studies to determine its physiological role. The electronic version of this dissertation is accessible from https://hdl.handle.net/1969.1/197123 |
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