This competing continuation application proposes the extension of mechanistic and structural analyses of flavoenzymes that function in key physiological and protective roles in microorganisms. Typical examples of flavoprotein disulfide reductases that serve this function include the sequence-related alkylhydroperoxide reductase, NADH peroxidase, glutathione reductase and the trypanosomal ortholog, trypanothione reductase, found uniquely in parasitic protozoans. With the determination of the genome sequence of the human pathogenic bacterium, Mycobacterium tuberculosis, the identification of putative flavin-containing enzymes using advanced sequence searching methods has become possible. Using these methods, a number of genes have been identified which are likely to encode flavoproteins. The subsequent cloning of these genes, and the expression and purification of the gene products has confirmed putative assignments based on primary sequence considerations. One example of the application of this method includes the identification of the flavin-containing disulfide reductase that catalyzes the reduction of the oxidized form of mycothiol; a recently discovered, structurally unique thiol present at high concentrations in mycobacteria and a limited number of additional bacterial species. It is likely that this enzyme is responsible for maintaining a reducing intracellular environment, and may contribute to the ability of the bacterium to survive the hostile oxidizing environment encountered in the macrophage phagolysosomal compartment. Two other sequence-related flavoenzymes have been identified, and the principal investigator will attempt to define the physiological function of these two proteins, determine their chemical mechanism and attempt to determine their three-dimensional structure. The principal investigator proposes that these latter enzymes play key roles in the oxidative stress management, survival of the organism and propagation of the infection. The experiments described are designed to assess these roles, and if found to be correct, could ultimately lead to the development of specific inhibitors with chemotherapeutic utility.
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