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Novel Carboxyl Proteinases : Structure, Function, and Evolution

新型羧基蛋白酶：结构、功能和进化

基本信息

批准号：
11694206
负责人：
ODA Kohei
金额：
$ 2.82万
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 (B).
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

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694206/
关键词：
carboxyl proteinase structure and function molecular evolution hereditary disease CLN2 subsite structure primary structure three-dimesional structure ペプスタチン

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 aminoacid 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). (l) 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101号CP（PCP）、黄单胞菌CP（XCP）、凝结芽孢杆菌CP（J-4）、新生芽孢杆菌CP（J-4）、枯草芽孢杆菌CP（J MN-32（嗜酸乳杆菌溶血素）和人来源的CLN 2。得到以下结果.一级结构我们成功地克隆了J-4蛋白酶基因。完全确定了上述“胃蛋白酶抑制剂不敏感CP”的所有一级序列。亚位点结构分析了阿莫拉溶血素的底物特异性。结果表明，该酶的S2亚位点很小，S2'亚位点主要由亲水性氨基酸残基和其他细菌来源的胃蛋白酶抑制剂不敏感CP组成.催化残基通过定点突变分析了CLN 2和4种细菌CP的推定催化氨基酸残基，特别是集中于Ser残基。结果表明，这些CP的催化残基主要由Asp、Glu和Ser残基组成，但仍存在一些问题.已阐明五氯苯酚的三维结构（《自然结构生物学》，2001年5月出版）。(l)PCP的三维结构与典型的丝氨酸蛋白酶枯草杆菌蛋白酶BPN '基本相似。(2)催化残基由Asp 84、Glu 80和Ser 287组成。目前还没有关于含有Ser残基的CP参与其催化作用的报道。因此，从遗传和结构上阐明这些CP是一种新型的蛋白酶。2000年11月10日，我们组织了一次名为“KIT International Conference on Carboxyl Proteinases and Their Inhibitors”的国际会议。