SQUALENE AND OXIDOSQUALENE CYCLASES

描述(逐字摘自摘要)：氧化十一烯环酶(OSCs)和 细菌性角鲨烯：霍普烯环化酶(SHCs)负责产生过量 80种不同的甾体和其他多环三萜结构。应用 基于选择性机理的不可逆缓蚀剂和光亲和标记 用于修饰真核OSCs和细菌SHCS的活性部位 构成了探索酶结构和结构的战略的关键 在这个酶家族中起作用。同质原生脊椎动物 氧化月桂烯：羊毛甾醇环化酶(OSLCs)，几种新的植物OSCs，a 从嗜热菌嗜酸杆菌重组SHC， 而一种可能来自人类结核分枝杆菌的SHC将是 用于亲和力标记、活性部位映射、三维 结构研究和抑制动力学。拟议的研究充分说明了 新型亲和探针的优势和利用分子生物学获得 重组酶、嵌合体和定点突变体以确定 与结合和催化有关的结构特征。 有六个具体目标。首先，未标记和氚标记的类似物， 抑制剂和光亲和标记将被合成并检测为 机械探头。第二，新的OSCs和SHCs，或者是从本地提纯的 来源或以重组形式表达，将被亲和标记，以便 绘制活性中心的重要催化区和模板区。第三, 将制备工程蛋白，包括OSC-SHC嵌合体和 定点突变体，以测试哪些氨基酸侧链对 循环酶活性。第四，获得大鼠功能表达的新途径 OSLC在真核表达系统中的应用将是今后的研究方向。第五，OSCS和SHCS 含有共价(或紧密)结合的抑制剂或底物类似物将 将被结晶，它们的三维结构将在 协作项目。最后，OSCs将与功能化角鲨烯一起使用 制备底物的类似物和用于研究的假定的抑制剂 新近克隆的可溶性酶环阿糖醇异构酶的活性部位 对植物中羊毛甾醇的生产至关重要。

DESCRIPTION (verbatim from the abstract): 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.