Engineering of flavonoid O-methyltransferase for a novel regioselectivity.
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.
Joe
Eun Ji
EJ
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea.
Kim
Bong-Gyu
BG
An
Byoung-Chan
BC
Chong
Youhoon
Y
Ahn
Joong-Hoon
JH
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
D005419:Flavonoids; D044949:Kaempferols; D050505:Mutant Proteins; D011994:Recombinant Proteins; C006552:kaempferol; D011794:Quercetin; D008780:Methyltransferases