STRUCTURE-FUNCTION OF ADP-RIBOSYL CYCLASE AND HOMOLOGS

ADP-核糖基环化酶及其同系物的结构-功能

• 批准号: 6498704
• 负责人: HONCHEUNG LEE
• 金额: $ 27.67万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498704
• 关键词: Aplysia; CD38 molecule; X ray crystallography; acid base balance; active sites; biological signal transduction; calcium flux; expression cloning; method development; protein structure function; site directed mutagenesis

ADP-ribosyl cyclase belongs to a family of homologous proteins important in mediating Ca2+ signaling in cells. The other members of the family include the differentiation antigens CD38 (on lymphocytes) and BST1/BP3 (on bone marrow cells). In addition to structural homology all members of the family share functional similarities as well. They all are enzymes catalyzing the synthesis of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), two structurally and functionally distinct Ca2+ messengers responsible for mobilizing different types of intracellular Ca2+ stores. The cADPR- and NAADP- sensitive Ca2+ stores have now been shown to be ubiquitously present in more than 40 different cell types from protozoart to human cells, indicating their general relevance. The cyclase family of enzymes is also catalytically novel. Contrary to normal enzymes which generally catalyze the conversion of one specific substrate to one product, the members of the family can use more than one substrate and convert them into structurally and functionally distinct products. Thus, the cyclase not only can cyclize NAD into cADPR, it can also use NADP as substrate and catalyze a base- exchange reaction with nicotinic acid, producing NAADP. Which catalytic path the cyclase takes is determined by pH. We have recently solved the crystal structure of the Aplysia cyclase and are in a position to characterize structure-function relationships of this novel multifunctional enzyme. Detailed knowledge of its catalytic mechanism is likely to have important ramifications in our understanding of Ca2 signaling mechanisms in cells. Specific aims are: l. To characterize the active sites of the Aplysia cyclase and human CD38. 2. To alter the active site by mutagenesis and assess the resulting changes in catalysis. 3. To determine the structure of the active site of the cyclase by X-ray crystallography. 4. To develop an inducible expression system of the cyclase for use in physiological studies.

ADP-核糖基环化酶属于在介导细胞中的Ca 2+信号中重要的同源蛋白质家族。该家族的其他成员包括分化抗原CD 38（在淋巴细胞上）和BST 1/BP 3（在骨髓细胞上）。除了结构上的同源性，该家族的所有成员也具有功能上的相似性。它们都是催化环ADP-核糖（cADPR）和烟酸腺嘌呤二核苷酸磷酸（NAADP）合成的酶，这两种结构和功能不同的Ca 2+信使负责动员不同类型的细胞内Ca 2+储存。cADPR-和NAADP-敏感的Ca 2+库现已显示普遍存在于从原生动物到人类细胞的40多种不同细胞类型中，表明它们的普遍相关性。酶的环化酶家族也是催化新颖的。与通常催化一种特定底物转化为一种产物的正常酶相反，该家族的成员可以使用一种以上的底物并将其转化为结构和功能不同的产物。因此，环化酶不仅可以将NAD环化成cADPR，它还可以使用NADP作为底物并催化与烟酸的碱交换反应，产生NAADP。环化酶的催化途径是由pH值决定的。我们最近已经解决了的Aaplasia环化酶的晶体结构，并能够表征这种新的多功能酶的结构-功能关系。其催化机制的详细知识很可能有重要的分支，在我们的理解细胞中的钙信号机制。具体目标是：l。表征失活菌环化酶和人CD 38的活性位点。2.通过诱变改变活性位点，并评估由此产生的催化变化。3.用X射线晶体学方法确定环化酶活性中心的结构。4.建立环化酶的诱导表达系统，用于生理学研究。