title: Engineering of flavonoid O-methyltransferase for a novel regioselectivity.
abstract: An O-methyltransferase isolated from poplar, POMT7, was identified as a flavone 7-O-methyltransferase. In order to generate a mutant of POMT-7 having a novel regioselectivity, we conducted an error-prone polymerase chain reaction. More than 100 mutants were screened and one of the mutants (POMT-M1) Asp257Gly, methylated the 3-hydroxyl group of flavonols in addition to 7-hydrdoxyl group. The mutation changed asparagine residue at the position of 257 into glycine. The kinetic parameters showed that the wild type POMT7 was better activity toward kaempferol and quercetin than the POMT7-M1. Using E. coli transformant expressing POMT7-M1, 58 microM of 3, 7-O-dimethylquercetin and 70 microM of 3, 7-O-dimethylkaempferol from 100 microM of corresponding substrate were synthesized successfully.
authors.affiliation: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea.
authors.forename: Eun Ji
authors.initials: EJ
authors.lastname: Joe
authors.forename: Bong-Gyu
authors.initials: BG
authors.lastname: Kim
authors.forename: Byoung-Chan
authors.initials: BC
authors.lastname: An
authors.forename: Youhoon
authors.initials: Y
authors.lastname: Chong
authors.forename: Joong-Hoon
authors.initials: JH
authors.lastname: Ahn
mesh_terms: D001665:Binding Sites; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism*; D044949:Kaempferols / Q000032:analysis / Q000737:chemistry; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D032107:Populus / Q000201:enzymology*; D015202:Protein Engineering / Q000379:methods*; D011794:Quercetin / Q000032:analysis / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D005419:Flavonoids; D044949:Kaempferols; D050505:Mutant Proteins; D011994:Recombinant Proteins; C006552:kaempferol; D011794:Quercetin; D008780:Methyltransferases