Gentisate 1,2-Dioxygenase from Xanthobacter polyaromaticivorans 127W
Gentisate 1,2-Dioxygenase from Xanthobacter polyaromaticivorans 127W
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DOI：
10.1271/bbb.60444
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发表时间：
2007-01
期刊：
影响因子：
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通讯作者：
S. Hirano;M. Morikawa;K. Takano;T. Imanaka;S. Kanaya
中科院分区：
文献类型：
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作者：
S. Hirano;M. Morikawa;K. Takano;T. Imanaka;S. Kanaya
假定的龙胆酸 1,2-双加氧酶是在来自 Xanthobacter polyaromaticivorans 127W 的二苯并噻吩降解基因簇 (dbd) 中编码的。推导的氨基酸序列与来自产碱假单胞菌（AAD49427，65％相同）、日本慢生根瘤菌（NP_766750，64％）和铜绿假单胞菌（ZP_00135722，54％）的龙胆酸双加氧酶显示出高度的序列相似性，并且与1-羟基-2-萘甲酸酯具有中等相似性来自诺卡氏菌属的双加氧酶。 KP7（BAA31235，33%）和来自水杨酸氧化假杆菌的水杨酸双加氧酶（AAQ91293，33%）。该酶 GDOxp 在大肠杆菌中异源产生并纯化至同质。 GDOxp 形成四聚体，并对 1,4-二羟基 2-萘甲酸酯和龙胆酸酯表现出高双加氧酶活性，表明异常广泛的底物特异性。 GDOxp在不利的温度和pH条件下很容易释放亚铁离子，成为无活性的单体蛋白。无活性的单体蛋白在加入亚铁离子后可以重建四聚体结构并以协同方式恢复酶活性。胰凝乳蛋白酶消化和蛋白质截短实验表明 N 末端区域对于 GDOxp 的四聚化很重要。
A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP_766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.