Characterization of the gallate dioxygenase gene:: Three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6
Characterization of the gallate dioxygenase gene:: Three distinct ring cleavage dioxygenases are involved in syringate degradation by Sphingomonas paucimobilis SYK-6
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DOI：
10.1128/jb.187.15.5067-5074.2005
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发表时间：
2005-08-01
影响因子：
3.2
通讯作者：
Fukuda, M
中科院分区：
文献类型：
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作者：
Kasai, D;Masai, E;Fukuda, M
少动鞘氨醇单胞菌SYK-6分别与四氢叶酸依赖性O-脱甲基酶LigM和德萨反应，将香草酸和胡萝卜酸转化为原儿茶酸（PCA）和3-O-甲基没食子酸（3 MGA）。PCA通过PCA 4，5-裂解途径进一步降解，而3 MGA通过三种不同的途径代谢，其中涉及PCA 4，5-双加氧酶（LigAB）、3 MGA 3，4-双加氧酶（DesZ）和3 MGA O-脱甲基酶（LigM）。在3 MGA O-去甲基化途径中，LigM将3 MGA转化为没食子酸，并且所得的没食子酸似乎被LigAB或DesZ以外的双加氧酶降解。在此，我们分离了没食子酸双加氧酶基因desB，它编码一个418个氨基酸的蛋白质，分子量为46，843 Da。与PCA 4，5-双加氧酶β和α亚基的序列分别具有40%和27%的同一性。纯化大肠杆菌中产生的DesB，并估计为同源二聚体（86 kDa）。DesB特异性地攻击没食子酸酯以生成作为反应产物的4-双羟甲基磺酸酯。测定没食子酸盐的Km和Vmax分别为66.9 +/- 9.3 μ M和42.7 +/- 2.4 U/mg。基于对缺乏参与儿茶酸降解的基因的各种SYK-6突变体的分析，我们得出结论：（i）所有三环裂解双加氧酶都参与儿茶酸催化，（ii）涉及LigM和DesB的途径在SYK-6在儿茶酸上的生长中起着特别重要的作用，以及（iii）DesB和LigAB参与没食子酸降解。
Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase beta and alpha subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K-m for gallate and the V-max were determined to be 66.9 +/- 9.3 mu M and 42.7 +/- 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.