This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ultra-high resolution structural studies of the flavoenzyme cholesterol oxidase (ChO) was carried out. ChO is a 55 kDa bifunctional flavoenzyme involved in the oxidation and isomerization of steroids that contain a 3-beta-hydroxyl group and a double bond at the C5 position of the steroid nucleus. The enzyme has been used clinically for the determination of serum cholesterol and most recently, due to the enzyme s ability to alter membrane structure, it has found applications as a potent larvicide. Moreover, the enzyme has also been proposed to be one of the virulence factors in Rhodococcus equi, an opportunistic pathogen in humans, associated with human immunodeficiency virus infection. In humans, Rhodococcus equi infection mainly affects the lungs with clinical pathological characteristics similar to pulmonary tuberculosis in immunocompromised patients. Cholesterol oxidase acts on eukaryotic membranes and its membrane damaging properties may be responsible for the observed cytotoxicity and macrophage destruction that accompanies Rhodococcal infection. Given the enzyme s prominent role in pathogenesis, we believe that a clear understanding of the substrate binding properties as well as the catalytic mechanism may aid in the development of inhibitors used as drugs to treat patients suffering from Rhodococcal infection. The purported channel for oxygen entry to the active site was studied using xenon incubation. Preliminary studies resulted in data sets extending to 0.85 (cell dimensions: a=51.267, b=73.213 c=63.070 alpha=90.0 beta=105.132 gamma=90). Although xenon was found to bind, it was not in the purported channel.
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