Oxidosqualene cyclases (OSCs) and bacterial squalene:hopene cyclases (SHCs) are responsible for generating over 80 different steroidal and other polycyclic triterpene structures. Applications of selective mechanism-based irreversible inhibitors and photoaffinity labels for modification of the active site of eukaryotic OSCs and bacterial SHCs constitute the linchpin of the strategy to explore enzyme structure and function within this enzyme family. Homogenous native vertebrate oxidosqualene:lanosterol cyclases (OSLCs), several novel plant OSCs, a recombinant SHC from a thermophilic bacteria Alicyclobacillus acidocaldarius, and a putative SHC from the human pathogen Mycobacterium tuberculosis will be used for the affinity labeling, active-site mapping, three-dimensional structural studies, and inhibition kinetics. The proposed research takes full advantage of the new affinity probes and harnesses molecular biology to obtain recombinant enzymes, chimeras, and site-directed mutants to determine the structural features involved in binding and catalysis. There are six specific goals. First, unlabeled and tritium-labeled analogs, inhibitors and photoaffinity labels will be synthesized and assayed as mechanistic probes. Second, new OSCs and SHCs, either purified from native sources or expressed in recombinant form, will be affinity labeled in order to map important catalytic and templating regions of the active sites. Third, engineered proteins will be prepared, including OSC-SHC chimeras and site-directed mutants, to test which amino acid side chains are important for cyclase activity. Fourth, new approaches to obtain functional expression of rat OSLC in eukaryotic expression systems will be pursued. Fifth, OSCs and SHCs containing covalently (or tightly) bound inhibitors or substrate analogs will be crystallized and their three-dimensional structures will be determined in collaborative projects. Finally, OSCs will be used with functionalized squalene analogs to prepare substrates and putative inhibitors for the study of the active site of cycloartenol isomerase, a recently-cloned soluble enzyme critical for lanosterol production in plants.
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