Dimethylsulfoniopropionate-Dependent Demethylase (DmdA) from Pelagibacter ubique and Silicibacter pomeroyi
Dimethylsulfoniopropionate-Dependent Demethylase (DmdA) from Pelagibacter ubique and Silicibacter pomeroyi
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DOI：
10.1128/jb.00770-08
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发表时间：
2008-12-01
影响因子：
3.2
通讯作者：
Whitman, William B.
中科院分区：
文献类型：
--
作者：
Reisch, Chris R.;Moran, Mary Ann;Whitman, William B.
藻类代谢产物二甲基磺基丙酸盐（DMSP）是海洋细菌的主要碳源和还原硫源。最近，负责DMSP的去甲基化的酶，指定DmdA，被确定，并发现同源物是常见的海洋浮游细菌细胞。采用阴离子交换、疏水性相互作用和羟基磷灰石层析三步纯化来自培养的海洋细菌Pelagibacter ubique HTCC 1062和Silicibacter pomeroyi DSS-3的重组DmdA蛋白。泛青霉酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的M-r为38，500。在非变性条件下，M-r为68，000，表明该酶可能是二聚体。纯化的酶对DMSP具有严格的底物特异性，与S. pomeroyi和P. ubique对甘氨酸甜菜碱、二甲基甘氨酸、甲基巯基丙酸酯、甲硫氨酸或二甲基磺基乙酸酯没有可检测的脱甲基酶活性。发现二甲基磺基丁酸酯和二甲基磺基戊酸酯的活性小于1%。DMSP的表观K（m）s为13.2 +/- 2.0和5.4 +/- 2.3 mM，P. ubique和S.分别为pomeroyi酶。在S.在pomeroyl DSS-3中，DMSP的表观Km为8.6 +/-1.2mM，与纯化的重组DmdA相似。在恒化器培养的S.这些结果表明，海洋浮游细菌可能会积极积累DMSP到有利于DMSP去甲基化活性接近最大速率的显著浓度。
The ubiquitous algal metabolite dimethylsulfoniopropionate ( DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria Pelagibacter ubique HTCC1062 and Silicibacter pomeroyi DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The P. ubique enzyme possessed an M-r on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the M-r was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both S. pomeroyi and P. ubique possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent K(m)s for DMSP were 13.2 +/- 2.0 and 5.4 +/- 2.3 mM for the P. ubique and S. pomeroyi enzymes, respectively. In cell extracts of S. pomeroyi DSS-3, the apparent K-m for DMSP was 8.6 +/- 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown S. pomeroyi DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.