M. tuberculosis (Mtb), a global health crisis and bioterrorism threat, is increasingly drug-resistant, but little new chemotherapy has emerged in decades. A fresh approach to chemotherapy is to target pathways essential for the pathogen to survive in its metabolic niche in host macrophages. This application is based on the hypothesis that Mtb requires 3 enzymes to survive under energy-poor, oxidative and nitrosative conditions in the phagosome: (1) Lipoamide dehydrogenase (Lpd) serves in pyruvate dehydrogenase and probably in branched chain ketoacid dehydrogenase as well as in peroxynitrite reductase /peroxidase and thus is key for net synthesis of acyl CoA's, the precursors of fatty acids, and for resistance to reactive nitrogen intermediates (RNI). (2) a-ketoglutarate (KG) decarboxylase (KDC) converts KG to succinic semialdehyde (SSA), replacing KG dehydrogenase, which Mtb lacks. SSA dehydrogenase converts SSA to succinate and may thereby connect the oxidative and reductive limbs of Mtb's citric acid cycle (CAC). KDC may therefore be important for Mtb's generation of energy, reducing equivalents, amino acids and heme. (3) Ultraviolet repair (Uvr) B, part of the nucleotide excision repair pathway, was found by saturation transposon mutagenesis to be essential for Mtb to survive RNI and to kill mice. KDC and UvrB lack human homologs, and Lpd's crystal structure shows key differences from the human enzyme. We will use allelic replacement to disrupt the 3 genes encoding these enzymes, or establish their essentiality; use conventional and novel combinatorial libraries to identify chemical inhibitors of each enzyme; analyze the crystal structures of Lpd and KDC with and without inhibitors; and assess these enzymes as potential targets for new chemotherapeutics. Antibiotics to date only target enzymes that synthesize protein, nucleic acids, cell walls and folate. The fundamental novelty of this work is to broaden the range of targets to include enzymes of intermediary metabolism and DNA repair.