The determination of the relationship between an enzyme's three-dimensional structure and its catalytic and physiological function continues to be the long-range objective of this research program. The expanding family of flavoprotein reductases represent an appropriate and unique selection for such investigations, since they are ubiquitously distributed, mechanistically intriguing and serve critical roles in the organisms in which they are present. Examples of these pivotal roles played by flavoprotein reductases include pro- and eukaryotic oxidative stress management involving alkyl hydroperoxide reductase, NADH peroxidase, glutathione reductase and its unique homologue found only in parasitic protozoal pathogens, trypanothione reductase. Members of the flavoprotein reductase family additionally serve to detoxify mercuric salts and assist in proper protein folding by catalyzing disulfide bond formation and isomerization. The amino acid sequences of many of these enzymes are known, and the three dimensional structure of six flavoprotein reductases have been determined at high resolution. The relationship between the structure and function of trypanothione reductase will be probed by a combination of kinetic, isotopic and mutagenic approaches. To understand the role played by flavoprotein reductases enabling pathogenic bacteria to withstand the large flux of reduced oxygen species inside macrophage, two new previously unidentified flavoproteins from mycobacteria, identified as a lipoamide dehydrogenase and an unprecedented disulfide reductase will be characterized. These enzymes will be purified, their kinetic and chemical mechanisms determined, the range of reactions catalyzed examined and the genes encoding the enzymes sequenced. These studies will provide a timely and relevant characterization of the role these enzymes play in the survival of these pathogens in an oxidatively stressful environment, and could ultimately lead to the development of specific inhibitors with chemotherapeutic utility.
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