Genetic and enzymatic analyses of C-P bond cleavage enzyme in bacteria

细菌 C-P 键裂解酶的遗传和酶学分析

• 批准号: 03660113
• 负责人: MURATA Kousaku
• 金额: $ 1.09万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03660113/
• 关键词: C-P bond; C-P bond cleavage enzyme; C-P hydrolase; C-P lyase; CーP結合; CーP結合解裂酵素; CーP結合加水分解酵素; CーPリア-ゼ

A direct carbon-phosphorus (C-P) bond is highly stable and resistant to chemical hydrolysis, thermal decomposition and photolysis. Therefore, biological cleavage of the C-P bond in natural habitats assumes importance as a mean to prevent the accumulation of phosphonates. Some bacterial cells are known to use various phosphonates for growth. However, all attempts to detect C-P bond cleavage activity in cell-free systems have resulted in failure, and the problem of how and by what means these bacteria can cleave the C-P bond in phosphonates remains unsolved. We have succeeded, for the first time, in the detection of C-P bond cleavage activity in cell-free extracts of a bacterium Enterobacter aerogenes, and isolated an enzyme responsible for the activity. The enzyme catalyzed the C-P bond cleavage and produced inorganic phosphate. Formation of alkane was not observed. The enzyme was then designated C-P hydrolase to descriminate from C-P lyase and phosphonatase. The enzyme required two proteins (E2 and E3) for activity. E2 was 650 kDa in molecular size. The function of E2 is to convert stable C-P bond into unstable one. E3 was a homodimer of a subunit with M.W. of 55 kDa. The function of the protein is to hydrolyze C-P bond being unstabled by E2 protein. The entire gene covering E2 and E3 structural regions was cloned from the organism and its structure was also analyzed.

直接碳磷（C-P）键高度稳定，耐化学水解、热分解和光解。因此，自然栖息地中 C-P 键的生物裂解作为防止膦酸盐积累的手段具有重要意义。已知一些细菌细胞使用各种膦酸盐进行生长。然而，所有在无细胞系统中检测 C-P 键裂解活性的尝试都以失败告终，并且这些细菌如何以及通过什么手段裂解磷酸盐中的 C-P 键的问题仍然悬而未决。我们首次成功检测了产气肠杆菌无细胞提取物中的 C-P 键裂解活性，并分离出了负责该活性的酶。该酶催化C-P键断裂并产生无机磷酸盐。没有观察到烷烃的形成。然后将该酶命名为C-P水解酶以区别C-P裂解酶和磷酸酶。该酶需要两种蛋白质（E2 和 E3）才能发挥活性。 E2的分子大小为650 kDa。 E2的作用是将稳定的C-P键转变为不稳定的C-P键。 E3 是亚基的同二聚体，分子量为 55 kDa。该蛋白的功能是水解因E2蛋白而不稳定的C-P键。从生物体中克隆了覆盖E2和E3结构区的整个基因，并分析了其结构。

项目成果

村田 幸作: "細菌のC-P結合解裂酵素" 日本生化学会誌. 64. 100-105 (1992)

Kosaku Murata：“细菌 C-P 键裂解酶”日本生化学会杂志 64. 100-105 (1992)。

村田 幸作: "細菌のCーP結合解裂酵素" 日本農芸化学会誌. 65. 1501-1504 (1991)

Kosaku Murata：“细菌 C-P 键裂解酶”日本农业化学学会杂志 65. 1501-1504 (1991)。

Kousaku Murata: "Detection of carbon-phosphorus lyase activity in cell extracts of Enterobacter aerogenes" Biochemical and Biophysical Research Communication. 157. 190-195 (1988)

Kousaku Murata：“产气肠杆菌细胞提取物中碳磷裂解酶活性的检测”生物化学和生物物理研究通讯。

Kousaku Murata, Noriko Higaki and Akira Kimura: "Carbon-phosphorus hydrolase: Some properties of the enzyme in cell extracts of enterobacter aerogenes" Agric. Biol. Chem.,. 53. 1225-1229 (1989)

Kousaku Murata、Noriko Higaki 和 Akira Kimura：“碳磷水解酶：产气肠杆菌细胞提取物中酶的一些特性”Agric。

村田 幸作: "細菌のCーP結合解裂酵素" 日本生化学会誌. (1992)

Kosaku Murata：“细菌 C-P 键裂解酶”日本生化学会杂志（1992 年）。