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.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets. An uncharted area of antibiotic discovery can be explored by inhibiting nutrient biosynthesis. Herein, we investigate the potential of inhibiting biotin biosynthesis in monotherapy and combination therapy approaches to treat multidrug-resistant Gram-negative pathogens. In chapter 2, we validate biotin biosynthesis as a viable target for Gram-negative pathogens. Historically, biotin biosynthesis was overlooked as a target in Gram-negative pathogens as there was no observed fitness cost associated with its inhibition in standard mouse infection models. We discovered traditional mouse models do not accurately represent the biotin levels in humans. We developed an innovative mouse model to account for this discrepancy, validating biotin biosynthesis as an antimicrobial target in the presence of human-mimicking levels of biotin. Exploiting this sensitivity, we show that an inhibitor of biotin biosynthesis, MAC13772, is efficacious against Acinetobacter baumannii in a systemic murine infection model. In chapter 3, we continue to investigate the potential of targeting biotin biosynthesis in a combination therapy approach. In this work, we identify the ability of MAC13772 to synergize with colistin exclusively against colistin-resistant pathogens. The first committed step of fatty acid biosynthesis requires biotin as a cofactor; therefore, it is indirectly inhibited through the action of MAC13772. We propose that the inhibition of fatty acid biosynthesis leads to changes in membrane fluidity and phospholipid composition, restoring colistin sensitivity. The combination of a fatty acid biosynthesis inhibitor and colistin proved superior to either treatment alone against mcr-1 expressing Klebsiella pneumoniae and colistin-resistant Escherichia coli murine infection models. Together, these data suggest that biotin biosynthesis is a robust antibiotic target for further development in monotherapy and combination therapy approaches. / Thesis / Doctor of Philosophy (PhD)

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Pichia pastoris, a methylotrophic yeast is widely used for recombinant protein production. It has a well characterized methanol utilization (MUT) pathway, the enzymes of which are induced when cells are cultured in the presence of methanol. In this study, we have identified an unannotated zinc finger protein, which was subsequently named ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) and characterized its function. ROP expression is induced in P. pastoris cells cultured in biotin depleted glucose ammonium medium as well as a medium containing methanol as the sole source of carbon. In glucose-abundant, biotin depleted cultures, ROP induces the expression of a number of genes including that encoding PEPCK. Interestingly, a strain in which the gene encoding ROP is deleted (ΔROP) exhibits biotin-independent growth. Based on a number of studies, it was proposed that the ability of ΔROP to grow in the absence of biotin is due to the activation of a pyruvate carboxylase-independent pathway of oxaloacetate biosynthesis. It was also proposed that PEPCK, which normally functions as a gluconeogenic enzyme, may act as an anaplerotic enzyme involved in the synthesis of oxaloacetate. ROP was shown to be a key regulator of methanol metabolism when P. pastoris cells are cultured in YPM medium containing yeast extract, peptone and methanol but not YNBM medium containing yeast nitrogen base and methanol. In P. pastoris cells cultured in YPM, ROP functions as a transcriptional repressor of genes encoding key enzymes of the methanol metabolism such as the alcohol oxidase I. (AOXI). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion while overexpression of ROP results in repression of AOXI and retardation of growth of P. pastoris cultured in YPM medium. Subcellular localization studies indicate that ROP translocates from cytosol to nucleus in cells cultured in YPM but not YNBM. To understand the mechanism of action of ROP, we examined its DNA-binding specificity. The DNA-binding domain of ROP shares 57% amino acid identity with that of Mxr1p, a master regulator of genes of methanol metabolism. We demonstrate that the DNA-binding specificity of ROP is similar to that of Mxr1p and both proteins compete with each other for binding to AOXI promoter sequences. Thus, transcriptional interference due to competition between Mxr1p and ROP for binding to the same promoter sequences is likely to be the mechanism by which ROP represses AOXI expression in vivo. Mxr1p and ROP are examples of transcription factors which exhibit the same DNA-binding specificity but regulate gene expression in an antagonistic fashion.