Identification, characterization, and cloning of a phosphonate monoester hydrolase from Burkholderia caryophilli PG2982
Identification, characterization, and cloning of a phosphonate monoester hydrolase from Burkholderia caryophilli PG2982
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DOI：
10.1074/jbc.271.42.25754
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发表时间：
1996-10-18
影响因子：
4.8
通讯作者：
Kishore, GM
中科院分区：
文献类型：
--
作者：
Dotson, SB;Smith, CE;Kishore, GM
据观察，草甘膦降解细菌丁香伯克霍尔德氏菌 PG2982 利用草甘膦甘油酯作为唯一的磷源。磷酸酯水解酶 (PEH) 将甘油基草甘膦水解为草甘膦被确定为矿化途径中的第一个代谢步骤，这一观察结果为磷酸酯水解酶活性提供了第一个生物学作用，纯化的 PEH 酶水解了几种磷酸酯，包括对硝基苯基苯基膦酸酯、β-萘基苯基膦酸酯和 5-溴-4-氯-3-吲哚基苯基膦酸酯，纯化的PEH还水解了一些磷酸二酯，包括对硝基苯基5'-胸苷单磷酸和对硝基苯基磷酰胆碱。确定的催化效率最高的底物是双-(对硝基苯基)磷酸酯，其 K-m 为 0.9 mM，k(cat) 为 6.2 x 10(2) min(-1)，表明该酶也可能作为磷酸二酯酶发挥作用。天然酶是 58 kDa 亚基的同四聚体，pI 为 4.2。该酶活性的最适pH值为9.0，并被Mn2+离子刺激14倍，但金属辅因子对于活性不是必需的，从纯化的PEH蛋白中获得N端和胰蛋白酶片段氨基酸序列，并用于克隆B. caryophilli PG2982基因，命名为pehA。讨论了该酶独特的底物特异性以及作为植物中新型条件致死基因的潜在用途。
The glyphosate degrading bacterium, Burkholderia caryophilli PG2982, was observed to utilize glyceryl glyphosate as a sole phosphorus source. The hydrolysis of glyceryl glyphosate to glyphosate by a phosphonate ester hydrolase (PEH) was identified as the first metabolic step in the mineralization pathway, This observation provides the first biological role for a phosphonate ester hydrolase activity, Purified PEH enzyme hydrolyzed several phosphonate esters including p-nitrophenyl phenylphosphonate, beta-naphthyl phenylphosphonate, and 5-bromo-4-chloro-3-indolyl phenylphosphonate, The purified PEH also hydrolyzed some phosphodiesters including p-nitrophenyl 5'-thymidine monophosphate and p-nitrophenyl phosphorylcholine. The most catalytically efficient substrate identified was bis-(p-nitrophenyl) phosphate with a K-m of 0.9 mM and a k(cat) of 6.2 x 10(2) min(-1), suggesting that the enzyme may also function in vice as a phosphodiesterase. The native enzyme was a homotetramer of 58-kDa subunits and exhibited a pi of 4.2. The enzyme activity had a pH activity optimum of 9.0 and was stimulated 14-fold by Mn2+ ions, but a metal cofactor was not essential for activity, N-terminal and tryptic fragment amino acid sequences were obtained from the purified PEH protein and used to clone the B. caryophilli PG2982 gene, designated pehA. The unique substrate specificity of the enzyme and potential use as a novel conditional lethal gene in plants are discussed.