Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6
Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6
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DOI：
10.1128/jb.186.15.4951-4959.2004
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发表时间：
2004-08-01
影响因子：
3.2
通讯作者：
Fukuda, M
中科院分区：
文献类型：
--
作者：
Kasai, D;Masai, E;Fukuda, M
少动鞘氨醇单胞菌SYK-6能够以各种木质素衍生的联芳基化合物作为唯一的碳源和能量源生长。这些化合物被该菌株中独特和特异的酶降解为香草酸和香草酸。香草酸和胡萝卜酸分别被四氢叶酸依赖性O-脱甲基酶转化为原儿茶酸（PCA）和3-O-甲基没食子酸（3 MGA）。以前的研究表明，这些化合物通过PCA 4，5-裂解途径进一步降解。然而，我们随后对编码PCA 4，5-双加氧酶0亚基的ligB插入突变体的分析表明，至少有一种替代途径参与3 MGA降解。在本研究中，我们分离的desZ基因，赋予3 MGA降解活性的大肠杆菌。推导的desZ的氨基酸序列与文献报道的一致。与11型外二醇双加氧酶具有20 - 43%的同一性。气相色谱-质谱分析表明，DesZ催化3 MGA的3，4-裂解。SYK-6中desZ和ligB的破坏导致双氧依赖性3 MGA转化活性的丧失，但所得突变体保留了在甜菜根上生长的能力。我们发现，当向反应混合物中加入四氢叶酸时，desZ ligB双突变体的细胞提取物能够将3 MGA转化为没食子酸，并且该突变体的细胞提取物降解没食子酸的程度与野生型相同。所有这些结果表明，阿糖胞苷酸通过多种3 MGA降解途径降解，其中ligAB，desZ，3XIGA O-脱甲基酶和没食子酸双加氧酶是参与者。
Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgailate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the 0 subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type 11 extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3XIGA O-demethylase, and gallate dioxygenase are participants.