cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures.
A cDNA encoding UDP-glucose: formononetin 7- O-glucosyltransferase, designated UGT73F1, was cloned from yeast extract-treated Glycyrrhiza echinata L. cell-suspension cultures using probes from Scutellaria baicalensis UDP-glucose: flavonoid 7- O-glucosyltransferase. The open reading frame of the UGT73F1 cDNA encodes a 441-amino-acid protein with a predicted molecular mass of 48.7 kDa. The deduced amino acid sequence showed that the protein is related to the stress-inducible glucosyltransferases. UGT73F1 mRNA was not detected in untreated G. echinata cultures but was transiently induced by treatment with yeast extract. Recombinant UGT73F1 was expressed as a histidine-tag fusion protein in Escherichia coli and purified to near homogeneity by nickel chelate chromatography. The purified recombinant enzyme was selective for isoflavonoid, formononetin and daidzein as substrates, while flavonoids and various tested non-flavonoid compounds were poor substrates.
Nagashima
Shigeyuki
S
School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, 108-8641 Tokyo, Japan.
Inagaki
Rieko
R
Kubo
Akiko
A
Hirotani
Masao
M
Yoshikawa
Takafumi
T
D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics*; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D006035:Glycyrrhiza / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D007529:Isoflavones / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics; D013379:Substrate Specificity
D018076:DNA, Complementary; D018744:DNA, Plant; D007529:Isoflavones; D010940:Plant Proteins; D005964:Glucosyltransferases