A UDP-Glucose:Isoflavone 7-O-Glucosyltransferase from the Roots of Soybean (Glycine max) Seedlings

Abstract

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-Oglucosyltransferase (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 UDPglucose to isoflavones to produce isoflavone 7-O-␤-D-glucosides (k cat ‫؍‬ 0.74 s ؊1 , K m for genistein ‫؍‬ 3.6 M, and K m for UDPglucose ‫؍‬ 190 M). 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.

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

“…The enzyme displayed high k cat values for quercetin and kaempferol, which were 10-to 100-fold higher than the values reported for many other UGTs at optimal pH (e.g., Noguchi et al, 2007).…”

“…The enzymatic reaction followed Michaelis-Menten kinetics, and the steady state kinetic parameters were determined at pH 9.1 and 308C as summarized in Table 2. The enzyme displayed high k cat values for quercetin and kaempferol, which were 10-to 100-fold higher than the values reported for many other UGTs at optimal pH (e.g., Noguchi et al, 2007).…”

“…The enzymatic reaction followed Michaelis-Menten kinetics, and the steady state kinetic parameters were determined at pH 9.1 and 308C as summarized in Table 2. The enzyme displayed high k cat values for quercetin and kaempferol, which were 10-to 100-fold higher than the values reported for many other UGTs at optimal pH (e.g., Noguchi et al, 2007).Vv GT6 catalyzed glucosyl transfer from UDP-Glc to flavonols. Remarkably, this enzyme also displayed a strong galactosyl transfer activity (126% of glucosyl transfer activity) from UDP-Gal to flavonols (Figure 2).…”

“…Each of these Vv GT genes was predicted to have a single intron. Phylogenetic analysis revealed that each of these genes is related to the GT1 family phylogenetic cluster (cluster I) of flavonoid 3-O-glycosyltransferases, as expected (Vogt and Jones, 2000;Sawada et al, 2005;Noguchi et al, 2007). Moreover, these genes formed a subcluster that was distinct from the Vv GT1-related subcluster ( Figure 1B).…”

“…The branches shown in red and black indicate the foreground and background lineages, respectively, of the UGT gene clusters on the basis of a branch-specific model with the dN/dS ratios of v 1 = 0.34 and v 0 = 0.22, as suggested by the maximum likelihood analysis (Yang, 2007 (Vogt and Jones, 2000) and a catalytic mechanism that inverts the anomeric configuration of a transferred sugar (Campbell et al, 1997;Wang, 2009). Phylogenetic analyses of plant family 1 UGTs show several phylogenetic clusters, which appear to be characterized by the regiospecificity of their glycosyl transfer to flavonoids (Vogt and Jones, 2000;Sawada et al, 2005;Noguchi et al, 2007). Moreover, the differentiation of the sugar donor specificity of UGTs appears to have occurred in a lineage-specific manner after establishment of general regiospecificity for the glycosyl acceptor (Yonekura-Sakakibara et al, 2007;Noguchi et al, 2009a;Ono et al, 2010).…”