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Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II
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Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II
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Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II
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Journal Article
Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II
2016
Overview
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 [CH
3
-Co
III
]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH
3
-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.
Publisher
Springer Berlin Heidelberg,Springer,Springer Nature B.V
Subject
/ Analysis
/ Bacteria
/ Biomedical and Life Sciences
/ Biotechnologically Relevant Enzymes and Proteins
/ Cloning
/ E coli
/ Enzymes
/ Eubacterium - isolation & purification
/ Gastrointestinal Tract - microbiology
/ genes
/ Genomes
/ Genomics
/ Humans
/ Microbial Genetics and Genomics
/ Oxidoreductases, O-Demethylating - genetics
/ Oxidoreductases, O-Demethylating - metabolism
/ Proteins
/ Recombinant Proteins - genetics
/ Recombinant Proteins - metabolism
/ Studies
