Structure-Function, and Molecular Evolution of NCL disease-related Novel Carboxyl Proteinases from Bacteria

与 NCL 疾病相关的细菌新型羧基蛋白酶的结构功能和分子进化

基本信息

批准号：
11660090
负责人：
ODA Kohei
金额：
$ 2.24万
依托单位：
Kyoto Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

A series of research for "pepstatin-insensitive CPs" was carried out for Pseudomonas sp. No.101 CP (PCP), Xanthomonas sp. CP (XCP), Bacillus coagulans CP (J-4), Bacillus novosp. MN-32 (kumanolysin), and CLN 2 of the human origin. The following results were obtained.1. Primary structures We succeeded for cloning of J-4 proteinase gene. All of the primary sequences of "pepstatin-insensitive CPs" as described above were completely determined.2. Subsite structures A substrate specificity of kumamolysin was analyzed. It was clarified that S2 subsite of the enzyme was very small, and S2' subsite was composed of hydrophilic amino acid residues as well as other pepstatin-insensitive CPs of bacterial origin.3. Catalytic residues The presumed catalytic amino acid residues of CLN2 and 4-types of bacterial CPs were analyzed by site-directed mutagenesis, especially focused on Ser residue. As a result, catalytic residues of these CPs were elucidated to be composed of Asp, Glu, and Ser residues, beside of some unclarified problems.4. Three-dimensional structure Three-dimensional structure of PCP was elucidated (Nature Structural Biology, in May, 2001 in press). (1) Three-dimensional structure of PCP was basically similar to a typical serine proteinase, subtilisin BPN'. (2) The catalytic residues were proved to be composed of Asp84, Glu80, and Ser287. There are no reports about CPs that have Ser residues involved in their catalysis. Therefore, these CPs were elucidated to be a new type of proteinase based on genetical and structural approach. We ranked these CPs as a fifth proteinase family, and named it as "serine-carboxyl proteinase".On Nov.10 in 2000 year, we organized an international conference named as "KIT International Conference on Carboxyl Proteinases and Their Inhibitors".

对假单胞菌sp进行了一系列针对“ pepstatin不敏感的CP”的研究。 No.101 CP（PCP），Xanthomonas sp。 CP（XCP），哥格拉氏芽孢杆菌CP（J-4），Novosp芽孢杆菌。 Mn-32（kumanolysin）和人类起源的CLN 2。获得以下结果1。我们成功地克隆了J-4蛋白酶基因的基本结构。如上所述，所有主要序列“对pepstatin不敏感的CP”均完全确定。2。亚铁结构分析了库马莫林蛋白的底物特异性。可以澄清的是，酶的S2 subsite非常小，S2'亚铁矿由亲水性氨基酸残基以及其他对细菌起源的pepstatin不敏感的CPS组成3。催化残基通过位置定向的诱变分析了CLN2的推测催化氨基酸残基和4型细菌CP，尤其是针对Ser残基的诱变。结果，这些CP的催化残基阐明了由ASP，GLU和SER残基组成，除了一些未稀有的问题之外。4。阐明了PCP的三维结构三维结构（自然结构生物学，2001年5月，印刷中）。（1）PCP的三维结构基本上类似于典型的丝氨酸蛋白酶枯草蛋白BPN'。（2）证明催化残基由ASP84，GLU80和SER287组成。没有关于CP的cps残基涉及其催化的报道。因此，基于遗传和结构方法，将这些CP阐明为一种新型蛋白酶。我们将这些CPS排名为第五蛋白酶家族，并将其命名为“丝氨酸 - 羧基蛋白酶”。在2000年11月10日，我们组织了一次国际会议，名为“ Kit国际羧基蛋白酶及其抑制剂”。