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: 6628838
• 负责人: HONCHEUNG LEE
• 金额: $ 28.48万
• 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/6628838
• 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-核糖基环酶属于一个对介导细胞中Ca2+信号的重要同源蛋白家族。该家族的其他成员包括分化抗原CD38（淋巴细胞）和BST1/BP3（在骨髓细胞上）。除结构同源性外，家庭的所有成员也具有功能相似之处。它们都是催化环环ADP-核糖（CADPR）和烟酸腺苷二核苷酸磷酸（NAADP）的合成的酶，这是两个结构和功能上不同的CA2+ Messenger，负责动员不同类型的细胞内CA2+存储。现在已经显示出，从原生动物到人类细胞的40多种不同的细胞类型中，CADPR-和NAADP敏感的Ca2+商店无处不在，表明它们的一般相关性。酶的环酶家族也是催化新颖的。与通常催化一种特定底物转换为一种产品的正常酶相反，该家族的成员可以使用多种底物并将其转换为结构和功能上不同的产品。因此，环化酶不仅可以将NAD循环到CADPR中，还可以将NADP用作底物并催化与烟酸产生NAADP的碱基交换反应。循环酶采取的哪种催化路径由pH确定。我们最近解决了Aplysia环酶的晶体结构，并且可以表征这种新型多功能酶的结构功能关系。对其催化机制的详细知识可能在我们对细胞中Ca2信号机制的理解中具有重要的后果。具体目标是：l。为了表征Aplysia Cyclase和Human CD38的活性位点。 2。通过诱变改变活性位点并评估催化的变化。 3。通过X射线晶体学确定循环酶的活性位点的结构。 4。开发一个可用于生理研究的环化酶的诱导型表达系统。