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. Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. When Mtb infects human alveolar macrophages, it inhibits normal phagosome-lysosome fusion, allowing Mtb to survive in a usually bactericidal environment. The mechanisms by which Mtb inhibits phagolysosome fusion and survives in vivo are poorly understood. Pioneering genetic studies identified Mtb genes required for fusion arrest. I hypothesize that two of these genes encode enzymes that produce a signaling lipid. Based on sequence analysis, these proteins may be a terpene cyclase and an enzyme that produces the linear substrate of the cyclase. I request beamtime at SSRL to determine the structures of these virulence enzymes. I grew crystals of the putative terpene cyclase and collected data to 2.8 ? resolution. A complex with the lipid product also will be analyzed to identify the product and define the mechanisms of binding and catalysis. These studies will establish the chemical reaction and enable development of inhibitors of this essential Mtb virulence factor.
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