Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II
Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II
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DOI：
10.1007/s00253-016-7626-1
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发表时间：
2016-11-01
影响因子：
5
通讯作者：
Yang, Dong-Hui
中科院分区：
文献类型：
--
作者：
Chen, Jia-Xing;Deng, Chao-Yin;Yang, Dong-Hui
在缺氧条件下，淤泥真杆菌ZL-Ⅱ可将开环异落叶松树脂酚（SECO）转化为脱甲基产物4，4 '-二羟基肠二醇（DHEND）。然而，反应级联仍然不清楚。在这里，O-脱甲基酶负责的转换进行了鉴定和表征。筛选、克隆了9个编码2种甲基转移酶-Ⅰ（MT-I）、2种类可啉蛋白（CP）、2种甲基转移酶-Ⅱ（MT-II）和3种激活酶（AE）的基因，并在大肠杆菌中表达。在9种预测酶中，有4种酶（ELI_2003（MT-I）、ELI_2004（CP）、ELI_2005（MT-II）和ELI_0370（AE））被证实是E. limosum ZL-Ⅱ.体外重建了完整的O-脱甲基酶反应体系。如所预期的，脱甲基产物3-去甲基-SECO和DHEND均产生。在反应过程中，ELI_2003（MT-I）首先催化甲基从SECO转移到ELI_2004（[Co-I]-CP）的类咕啉，生成脱甲基产物和[CH 3-Co-III]-CP;然后ELI_2005（MT-II）介导甲基从[CH 3-Co-III]-CP转移到四氢叶酸，生成甲基四氢叶酸和[Co-I]-CP。由于[Co-II]/[Co-I]的低氧化还原电位，[Co-I]-CP在体外立即被氧化为[Co-II]-CP，并且ELI_0370（AE）负责催化[Co-II]-CP还原为其活性形式[Co-I]-CP。随后使用分子建模结合定点诱变确定ELI_2003、ELI_2005和ELI_0370中的活性位点残基。据我们所知，这是第一个从E. limosum ZL-II的合成方法，为相关生物活性物质的人工合成奠定了基础。
Eubacterium limosum ZL-II was described to convert secoisolariciresinol (SECO) to its demethylating product 4,4'-dihydroxyenterodiol (DHEND) under anoxic conditions. However, the reaction cascade remains unclear. Here, the O-demethylase being responsible for the conversion was identified and characterized. Nine genes encoding two methyltransferase-Is (MT-I), two corrinoid proteins (CP), two methyltransferase-IIs (MT-II), and three activating enzymes (AE) were screened, cloned, and expressed in Escherichia coli. Four of the nine predicted enzymes, including ELI_2003 (MT-I), ELI_2004 (CP), ELI_2005 (MT-II), and ELI_0370 (AE), were confirmed to constitute the O-demethylase in E. limosum ZL-II. The complete O-demethylase (combining the four components) reaction system was reconstructed in vitro. As expected, the demethylating products 3-demethyl-SECO and DHEND were both produced. During the reaction process, ELI_2003 (MT-I) initially catalyzed the transfer of methyl group from SECO to the corrinoid of ELI_2004 ([Co-I]-CP), yielding demethylating products and [CH3-Co-III]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH3-Co-III]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [Co-I]-CP. Due to the low redox potential of [Co-II]/[Co-I], [Co-I]-CP was oxidized to [Co-II]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [Co-II]-CP to its active form [Co-I]-CP. The active-site residues in ELI_2003, ELI_2005, and ELI_0370 were subsequently determined using molecular modeling combined with site-directed mutagenesis. To our knowledge, this is the first study on the identification and characterization of a four-component O-demethylase from E. limosum ZL-II, which will facilitate the development of method to artificial synthesis of related bioactive chemicals.