Cloning and characterization of a novel O-methyltransferase from Flammulina velutipes that catalyzes methylation of pyrocatechol and pyrogallol structures in polyphenols

Abstract

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.