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对从类产碱假单胞菌JS45克隆的pNBZ139中的羟基氨基苯突变酶的表征。一种高度相关的SDS稳定酶,催化羟基的分子内转移。

Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45. A highly associated SDS-stable enzyme catalyzing an intramolecular transfer of hydroxy groups.

作者信息

He Z, Nadeau L J, Spain J C

机构信息

Air Force Research Laboratory, Tyndall Air Force Base, FL 32403, USA.

出版信息

Eur J Biochem. 2000 Feb;267(4):1110-6. doi: 10.1046/j.1432-1327.2000.01107.x.

Abstract

Hydroxylaminobenzene mutase is the enzyme that converts intermediates formed during initial steps in the degradation of nitrobenzene to a novel ring-fission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase catalyzes a rearrangement of hydroxylaminobenzene to 2-aminophenol. The mechanism of the reactions and the properties of the enzymes are unknown. In crude extracts, the hydroxylaminobenzene mutase was stable at SDS concentrations as high as 2%. A procedure including Hitrap-SP, Hitrap-Q and Cu(II)-chelating chromatography was used to partially purify the enzyme from an Escherichia coli clone. The partially purified enzyme was eluted in the void volume of a Superose-12 gel-filtration column even in the presence of 0.05% SDS in 25 mM Tris/HCl buffer, which indicated that it was highly associated. When the enzymatic conversion of hydroxylaminobenzene to 2-aminophenol was carried out in 18O-labeled water, the product did not contain 18O, as determined by GC-MS. The results indicate that the reaction proceeded by intramolecular transfer of the hydroxy group from the nitrogen to the C-2 position of the ring. The mechanism is clearly different from the intermolecular transfer of the hydroxy group in the non-enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate.

摘要

羟氨基苯变位酶是一种能将假产碱假单胞菌JS45中硝基苯降解初始步骤形成的中间体转化为一种新的环裂解低分子量途径的酶。该变位酶催化羟氨基苯重排为2-氨基苯酚。反应机制和酶的性质尚不清楚。在粗提取物中,羟氨基苯变位酶在高达2%的SDS浓度下仍保持稳定。采用包括Hitrap-SP、Hitrap-Q和Cu(II)螯合色谱在内的方法从大肠杆菌克隆中部分纯化该酶。即使在25 mM Tris/HCl缓冲液中存在0.05% SDS的情况下,部分纯化的酶也在Superose-12凝胶过滤柱的空体积中洗脱,这表明它高度聚集。当在18O标记的水中进行羟氨基苯向2-氨基苯酚的酶促转化时,通过GC-MS测定,产物中不含18O。结果表明,该反应是通过羟基从氮原子向环的C-2位置的分子内转移进行的。该机制明显不同于羟氨基苯非酶促重排为4-氨基苯酚以及分支酸酶促羟化变为异分支酸过程中羟基的分子间转移。

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