Structure/function analysis of abundance and transmission mechanism of genetic information invlolved in the biosynthesis of multi-catalytic site enzyme

多催化位点酶生物合成涉及的遗传信息丰度及传递机制的结构/功能分析

• 批准号: 11460039
• 负责人: MURATA Kousaku
• 金额: $ 9.15万
• 依托单位: KYOTO UNIVERSITY
• 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 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460039/
• 关键词: sphingomonas; alginate lyase; autoprocessing; protease; x-ray crystallography; β-elimination mechanism; 遺伝情報多重性; プロテオーム; 遺伝情報量; 多触媒中心酵素; 翻訳後修飾; X線結晶構造解析; アルギン酸; Shingomonas属細菌; プロテオーム解析

The intracellular alginate lyases I,II, and III are coded by one gene and produced through sequential post-translational reactions from a common pre-cursor protein Po : Po → I + 5 kDa peptide, I → II +III. The protease-like activity of I is completely inhibited by EDTA or O- phnonthroline, thus indicates that the I is a multi- catalytic site enzyme currying II, III, and protease. The protease site on I is thought to be created through the excision of N-terminal 5 kDa peptide from Po, since Po has no protease activity. An important biological problem is then raised as to how the protease site is generated and how / where the information is described in the base and amino acid sequences. The clarification of these addresses may lead to the postulation of a novel theory concerning the abundance of information contained in a gene. To solve the problems, the knowleges on the structure / function relationship of alginate lyases I, II, and III, as well as their precursor protein Po. Among these, the structure / function of III was completely analyzed through X-ray crystallography and detailed lyase reaction mechanism was clarified. Especially, a novel β-elimination reaction mechanism involving Tyr residue, but not His, was constructed with an emphasis of Low-Barrier Hydrogen Bond Theory.

胞内藻酸盐裂解酶I、II和III由一个基因编码，并通过顺序翻译后反应从共同的前体蛋白Po产生：Po → I + 5 kDa肽，I → II +III。酶Ⅰ的蛋白酶样活性可被EDTA或邻菲咯啉完全抑制，表明酶Ⅰ是一个多催化位点酶，催化Ⅱ、Ⅲ和蛋白酶。I上的蛋白酶位点被认为是通过从Po切除N-末端5 kDa肽而产生的，因为Po没有蛋白酶活性。然后提出了一个重要的生物学问题，即蛋白酶位点是如何产生的，以及这些信息是如何/在哪里在碱基和氨基酸序列中描述的。这些地址的澄清可能会导致一个新的理论关于丰富的信息包含在一个基因的假设。为了解决这些问题，需要了解海藻酸裂解酶I、II和III及其前体蛋白Po的结构/功能关系。其中，III的结构/功能通过X-射线晶体学进行了全面分析，并阐明了详细的裂解酶反应机理。特别地，以低垒氢键理论为重点，构建了一个新的涉及Tyr残基而非His残基的β-消除反应机理。

项目成果

Keiko Momma: "Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1 at 2.0A resolution"J. Mol. Biol.. 316(5). 1061-1069 (2002)

Keiko Momma：“AlgQ2 的晶体结构，AlgQ2 是鞘氨醇单胞菌 A1 的一种大分子（藻酸盐）结合蛋白，分辨率为 2.0A”J。

Hye-Jin Yoon: "Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0A resolution"J. Mol. Biol.. 307. 9-16 (2001)

Hye-Jin Yoon：“2.0A 分辨率下藻酸裂解酶 A1-III 与三糖产物复合的晶体结构”J。

Hye-Jin Yoon: "Effect of His192 mutation on the activity of alginate lyase A1-III from Sphingomonas species A1"J. Microbiol. Biotechnol.. 11(1). 118-123 (2001)

Hye-Jin Yoon：“His192 突变对鞘氨醇单胞菌 A1 种藻酸盐裂解酶 A1-III 活性的影响”J。

Yumiko Mishima: "Super-channel in bacteria macromolecule uptake and depolymerization systems of Sphingomonas sp A1 with special cell surface structure"Biotechnology & Genetic Engineering Reviews. (in press). (2002)

三岛由美子：“具有特殊细胞表面结构的鞘氨醇单胞菌A1的细菌大分子摄取和解聚系统中的超级通道”生物技术

Wataru Hashimoto: "Super-channel in bacteria : Function and structure of a macromolecule import system mediated by a pit-dependent ABC transporter"Biosci. Biotechnol. Biochem.. 65(9). 1949-1956 (2001)

Wataru Hashimoto：“细菌中的超级通道：由凹坑依赖性 ABC 转运蛋白介导的大分子输入系统的功能和结构”Biosci。