A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis.
Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-beta-D-glucosides (k(cat) = 0.74 s(-1), K(m) for genistein = 3.6 microM, and K(m) for UDP-glucose = 190 microM). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1.
Noguchi
Akio
A
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11, Sendai 980-8579, Japan.
Saito
Atsushi
A
Homma
Yu
Y
Nakao
Masahiro
M
Sasaki
Nobuhiro
N
Nishino
Tokuzo
T
Takahashi
Seiji
S
Nakayama
Toru
T
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D005964:Glucosyltransferases / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D029902:Medicago / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D018517:Plant Roots / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000378:metabolism*; D027843:Vitis / Q000378:metabolism
D005964:Glucosyltransferases; C032691:UDP-glucose-isoflavone 7-O-glucosyltransferase