Mycobacterium tuberculosis (Mtb) utilizes different metabolic pathways for its survival during infection. Enzymes of these pathways are often targets for antibiotic development. Genetic studies indicate the importance of the serine and biotin biosynthetic pathways for Mtb survival. In this study, enzymes from these pathways were characterized using X-ray crystallographic and biochemical studies. D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step of phosphorylated serine biosynthesis. In comparison to other forms of PGDH, the Mtb enzyme has an insertion near its C-terminus. This insertion results in two different conformations of the subunits in the tetramer, leading to two different environments for cofactor binding. This intervening domain might provide a second binding site for hydroxypyruvic acid phosphate (HPAP) that is responsible for substrate inhibition. Analysis of the HPAP-bound Mtb PGDH active site reveals the residues (Arg52, Arg131, and Arg233) involved in substrate interaction and provides insights into a possible enzyme mechanism. Mtb PGDH is feedback inhibited by the end product Lserine. Examination of the serine-bound PGDH structure elucidates the key players (Tyr461, Asp463, and Asn481) involved in this allosteric inhibition, as well as the resultant conformational changes at the regulatory domain interface. Preliminary biochemical studies of the first enzyme in Mtb biotin biosynthesis, 7- keto-8-aminopelargonic acid (KAPA) synthase show that it exists as a dimer in solution and has higher substrate affinity than the E. coli enzyme. The second enzyme, 7, 8-diaminopelargonic acid synthase (DAPAS) uses Sadenosyl methionine and KAPA as substrates in a bi-bi ping-pong mechanism. A comparison of the substrate analog sinefungin-bound Mtb DAPAS structure with a KAPA-bound DAPAS model provides a basis for the dual-substrate recognition. Tyr25 is a key player in the substrate specificity in DAPAS; this was confirmed by mutation studies. In certain Bacillus species, a Phe replaces this Tyr. The KAPA-bound B. subtilis DAPAS structure shows an alteration in the KAPA binding mode. Substrate and product bound structures of the third enzyme, dethiobiotin synthetase (DTBS) in Mtb reveal the important residues involved in its catalysis and provide framework for a possible enzyme mechanism. Comparison to the DTBS structures from E. coli and H. pylori reveals differences in local conformations.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Description based on print version record. Includes bibliographical references (156-162).

Induced and activated as part of the innate immune response, the first line of defence against bacterial or viral infections, Inhibitor of Kappa-B Kinase ε (IKKε) triggers NF-κB and IFNβ signalling. Whilst not expressed at basal levels in healthy cells and tissue, the kinase is overexpressed in roughly 30% of human breast cancer cases, driving oncogenesis through aberrant activation of NF-κB. The impracticality of therapeutic targeting of NF-κB for cancer treatment has led to a requirement for greater understanding of IKKε's oncogenic potential to treat tumours driven by the kinase. Considering that IKKε alters cellular metabolism in dendritic cells, promoting aerobic glycolysis akin to the metabolic phenotype observed in cancer, it was hypothesised that the kinase would play a similar role in breast cancer. Using a Flp-In 293 model of IKKε induction and suppressing IKKε expression in a panel of breast cancer cell lines using siRNA, IKKε-dependent changes in cellular metabolism were characterised using labelled metabolite analysis. IKKε was found to induce serine biosynthesis, an important pathway in breast cancer development that supports glutamine-fuelling of the TCA cycle and contributes to one carbon metabolism to maintain redox balance. Promotion of serine biosynthesis occurred via a dual mechanism. Firstly, PSAT1, the second enzyme of the pathway, was found to be phosphorylated in an IKKε-dependent manner, promoting protein stabilisation. Secondly, an IKKε-dependent transcriptional upregulation of all three serine biosynthesis enzymes, PHGDH, PSAT1 and PSPH, was observed, induced by the inhibition of mitochondrial activity and the subsequent induction of ATF4-mediated mitochondria-to-nucleus retrograde signalling. These data demonstrate a previously uncharacterised mechanism of metabolic regulation by IKKε and highlight new potential therapeutic targets for the treatment of IKKε-driven breast cancer in the form of the enzymes of the serine biosynthesis pathway.