Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440 -: The prototype of a new subgroup of extradiol dioxygenases
Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440 -: The prototype of a new subgroup of extradiol dioxygenases
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DOI：
10.1074/jbc.m502585200
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发表时间：
2005-10-21
影响因子：
4.8
通讯作者：
Díaz, E
中科院分区：
文献类型：
--
作者：
Nogales, J;Canales, A;Díaz, E
在这项工作中，我们鉴定了恶臭假单胞菌 KT2440 的 galA 基因产物，这是一种环裂解双加氧酶，专门作用于没食子酸以产生 4-草酰中康酸。该蛋白质是由三个相同的 47.6 kDa（419 个氨基酸）亚基组成的三聚体，以 Fe2+ 作为主要辅因子。没食子酸双加氧酶在pH 7.0时表现出最大活性，没食子酸的K-m和V-max值分别为144μM和53.2μmol/min/mg蛋白质。系统发育研究表明，来自恶臭假单胞菌 KT2440 的没食子酸双加氧酶是 II 型外二醇双加氧酶新亚群的原型，该亚群与原儿茶酸 4,5-双加氧酶具有共同的祖先，并且其双结构域结构可能是由后者大小亚基的融合进化而来的。通过与来自少动鞘氨醇单胞菌 SYK-的原儿茶酸 4,5-双加氧酶的大 (LigB) 和小 (LigA) 亚基的晶体结构进行比较，生成了来自恶臭假单胞菌 KT2440 的没食子酸双加氧酶的 N 端结构域（残基 1 - 281）和 C 端结构域（残基 294 - 420）的三维模型。 6.当恶臭假单胞菌KT2440细胞在没食子酸酯存在下生长时，galA基因的表达被特异性诱导。恶臭假单胞菌 KT2440 galA 突变菌株无法使用没食子酸作为唯一碳源，并且不显示没食子酸双加氧酶活性，表明 GalA 蛋白是该细菌中参与没食子酸裂解的唯一双加氧酶。这项工作指出存在一种致力于没食子酸分解代谢的新途径，但在典型恶臭假单胞菌 KT2440 菌株中该途径仍然未知。
In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring- cleavage dioxygenase that acts specifically on gallate to produce 4- oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa ( 419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the K-m and V-max values for gallate were 144 mu M and 53.2 mu mol/ min/ mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5- dioxygenases and whose two- domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three- dimensional model for the N- terminal domain ( residues 1 - 281) and C- terminal domain ( residues 294 - 420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large ( LigB) and small ( LigA) subunits of the protocatechuate 4,5- dioxygenase from Sphingomonas paucimobilis SYK- 6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.