title: Cloning and characterization of a novel O-methyltransferase from Flammulina velutipes that catalyzes methylation of pyrocatechol and pyrogallol structures in polyphenols.
abstract: A novel O-methyltransferase gene was isolated from Flammulina velutipes. The isolated full-length cDNA was composed of a 690-nucleotide open reading frame encoding 230 amino acids. A database search revealed that the deduced amino acid sequence was similar to those of other O-methyltransferases; the highest identity was only 61.8% with Laccaria bicolor. The recombinant enzyme was expressed by Escherichia coli. BL21 (DE3) was assessed for its ability to methylate (-)-epigallocatechin-3-O-gallate (EGCG). LC-TOF-MS and NMR revealed that the enzyme produced five kinds of O-methylated EGCGs: (-)-epigallocatechin-3-O-(3-O-methyl)gallate, (-)-epigallocatechin-3-O-(4-O-methyl)gallate, (-)-epigallocatechin-3-O-(3,4-O-dimethyl)gallate, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)gallate, and (-)-4'-O-methylepigallocatechin-3-O-(3,5-O-dimethyl)gallate. The substrate specificity of the enzyme for 20 kinds of polyphenols was assessed using the crude recombinant enzyme of O-methyltransferase. This enzyme introduced methyl group(s) into polyphenols with pyrocatechol and pyrogallol structures.
authors.affiliation: a Research & Development-Production Headquarters, Asahi Breweries Limited , Moriya-shi , Ibaraki , Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Kirita
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Tagashira
authors.forename: Tomomasa
authors.initials: T
authors.lastname: Kanda
authors.forename: Mari
authors.initials: M
authors.lastname: Maeda-Yamamoto
mesh_terms: D000595:Amino Acid Sequence; D002392:Catechin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000378:metabolism; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D055393:Flammulina / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D059808:Polyphenols / Q000737:chemistry / Q000378:metabolism; D011748:Pyrogallol / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002396:Catechols; D005656:Fungal Proteins; D059808:Polyphenols; D011994:Recombinant Proteins; D011748:Pyrogallol; D005707:Gallic Acid; D002392:Catechin; C045651:epigallocatechin gallate; D008780:Methyltransferases; D002394:Catechol O-Methyltransferase; C573278:epigallocatechin-3-O-(3''-O-methyl)-gallate
keywords: Flammulina velutipes; O-methylated EGCGs; O-methylated polyphenols; O-methyltransferase