Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II

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• 作者：Jia-Xing Chen ; Chao-Yin Deng ; Ying-Tao Zhang…
• 关键词：O ; demethylase ; Eubacterium limosum ZL ; II ; Secoisolariciresinol ; Site ; directed mutagenesis
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：100
• 期：21
• 页码：9111-9124
• 全文大小：2,189 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614
• 卷排序：100

文摘

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 ([CoI]-CP), yielding demethylating products and [CH₃-CoIII]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH₃-CoIII]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [CoI]-CP. Due to the low redox potential of [CoII]/[CoI], [CoI]-CP was oxidized to [CoII]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [CoII]-CP to its active form [CoI]-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.