PURIFICATION AND CHARACTERIZATION OF THE FLAVOPROTEIN TRYPTOPHAN 2-MONOOXYGENASE EXPRESSED AT HIGH-LEVELS IN ESCHERICHIA-COLI
PURIFICATION AND CHARACTERIZATION OF THE FLAVOPROTEIN TRYPTOPHAN 2-MONOOXYGENASE EXPRESSED AT HIGH-LEVELS IN ESCHERICHIA-COLI
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DOI：
10.1006/abbi.1995.1034
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发表时间：
1995-01-10
影响因子：
3.9
通讯作者：
FITZPATRICK, PF
中科院分区：
文献类型：
--
作者：
EMANUELE, JJ;HEASLEY, CJ;FITZPATRICK, PF
来自萨瓦斯坦假单胞菌的色氨酸 2-单加氧酶是一种黄素蛋白，可催化色氨酸形成吲哚乙酰胺。这是在植物感染过程中形成吲哚乙酸以及随后由该细菌和其他细菌形成瘿的两步途径中的第一步。该酶已在大肠杆菌中高水平表达，并开发了一种纯化程序，可产生微摩尔量的蛋白质。纯化的酶含有紧密结合的吲哚乙酰胺；已开发出一种用 20% 甲醇透析的方法，用于去除吲哚乙酰胺而不显着损失酶活性，具有大疏水侧链的氨基酸是最好的底物； N-取代的苯丙氨酸也将充当底物，N-乙基马来酰亚胺、甲硫醇磺酸甲酯和焦碳酸二乙酯充当活性位点定向试剂，与酶活性位点处或附近的组氨酸和半胱氨酸一致，乙烯基甘氨酸部分地使酶失活，而炔丙基甘氨酸则没有作用。 (C) 1995 学术出版社
Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprotein which catalyzes the formation of indoleacetamide from tryptophan. This is the first step in a two-step pathway for the formation of indoleacetic acid during infection of plants and subsequent gall formation by this and other bacteria. The enzyme has been expressed in Escherichia coli at high levels, and a purification procedure has been developed which generates micromolar amounts of protein, The purified enzyme contains tightly bound indoleacetamide; a method involving dialysis against 20% methanol has been developed for removing the indoleacetamide without significant loss of enzyme activity, Amino acids with large hydrophobic side chains are the best substrates; N-substituted phenylalanines will also act as substrates, N-ethylmaleimide, methyl methanethiol-sulfonate, and diethylpyrocarbonate act as active site-directed reagents, consistent with a histidine and a cysteine at or near the enzyme active site, Vinylglycine partially inactivates the enzyme, while propargylglycine has no effect. (C) 1995 Academic Press,Inc.