title: Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II.
abstract: 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.
authors.affiliation: Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China.
authors.forename: Jia-Xing
authors.initials: JX
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Chao-Yin
authors.initials: CY
authors.lastname: Deng
authors.affiliation: Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China.
authors.forename: Ying-Tao
authors.initials: YT
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China.
authors.forename: Zhen-Ming
authors.initials: ZM
authors.lastname: Liu
authors.affiliation: Department of Immunology, School of Basic Medical Sciences, Peking University, Beijing, 100191, China.
authors.forename: Ping-Zhang
authors.initials: PZ
authors.lastname: Wang
authors.affiliation: Genomics Research Center, Harbin Medical University, Harbin, 150081, China.
authors.forename: Shu-Lin
authors.initials: SL
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. qianw@sun.im.ac.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Qian
authors.affiliation: Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China. ydhui@bjmu.edu.cn.
authors.forename: Dong-Hui
authors.initials: DH
authors.lastname: Yang
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D005051:Eubacterium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D041981:Gastrointestinal Tract / Q000382:microbiology; D006801:Humans; D010090:Oxidoreductases, O-Demethylating / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010090:Oxidoreductases, O-Demethylating
keywords: Eubacterium limosum ZL-II; O-demethylase; Secoisolariciresinol; Site-directed mutagenesis