Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

He, Xiping; Wang, Xiaoqiang; Dixon, Richard A.

doi:10.1074/jbc.m605767200

Cited by 113 publications

(124 citation statements)

References 38 publications

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“…Other UGTs are highly specific for a single acceptor (Fukuchi-Mizutani et al, 2003;Kramer et al, 2003). An emerging picture is that UGTs show regiospecificity toward the part of the substrate molecule harboring the site of glycosylation (Vogt et al, 1997;Kramer et al, 2003;Lim et al, 2003bRichman et al, 2005;He et al, 2006). This would be in accordance with the broad acceptor specificity of some UGTs, as the presence of a specific structural domain to be glycosylated can be presented on otherwise differing molecules.…”

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confidence: 48%

“…Biochemical studies seem to support regiospecificity as a key parameter for UGT substrate specificity. At the same time it is also evident that the presence of a specific structural domain will not by itself assure activity, but should be considered in conjunction with the overall structure of the aglycone (Richman et al, 2005;He et al, 2006;Kogawa et al, 2007;Modolo et al, 2007). Several studies of biochemically described UGTs show some conservation of regiospecificity within phylogenetic groups (Vogt and Jones, 2000;Lim et al, 2003a;Morita et al, 2005;Yonekura-Sakakibara et al, 2007;Caputi et al, 2008).…”

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confidence: 99%

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Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

et al. 2008

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The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidinderived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the substrate specificity of isolated wild-type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 2.5% of wild-type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDPglucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild-type enzyme activity toward delphinidin-3-O-glucoside was only 5% to 10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B ring were introduced to increase the activity toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity.

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confidence: 48%

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confidence: 99%

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

et al. 2008

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“…For example, His366, Asp374 and Asp390 are predicted to play an important role as a catalytic domain of UGTs, based on site-directed mutagenesis and computer modeling experiments [17,18,22]. Recent investigations on crystal structures of glucosyltransferases from Medicago truncatula [19,20] and Vitis vinifera [21] confirmed that these amino acids are key players in UDP-glucose recognition. However, potential roles in the overall glucose transfer reaction of less well-conserved amino acids within an individual UGT have attracted little attention.…”

Section: Site-directed Mutagenesis Of Caugt2mentioning

confidence: 99%

“…Investigation of the 3D-structures of betanidin 5-O-glucosyltransferase (B5GT) from Dorotheanthus bellidiformis [17] and cyanohydrin glucosyltransferase from Sorghum bicolor [18] by homology modeling, and of isoflavonoid 3 0 -O-glucosyltransferase from Medicago truncatula [19,20] and flavonoid 3-O-glucosyltransferase from Vitis vinifera [21] by X-ray crystallography, revealed the role of specific conserved amino acid residues in the PSPG-box that constitute the donor-sugar binding pockets. However, the roles of less well conserved amino acids within the motif that may determine the characteristics unique to particular enzymes such as substrate recognition and catalytic potential have been less closely examined.…”

Section: Introductionmentioning

confidence: 99%

A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

2007

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Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site-directed mutagenesis was carried out. Exchange of the PSPG-box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG-box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site-directed mutagenesis revealed that the size of the amino acid side-chain in that particular site is critical to the catalytic activity of CaUGT2.

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“…These PSPGs are characterized by a unique well conserved sequence of ϳ45 amino acid residues (called a PSPG box (14)) and a catalytic mechanism leading to an inversion of the anomeric configuration of transferred sugar (12). Recent crystallographic studies of PSPGs from Medicago truncatula (UGT71G1) (20) and Vitis vinifera (VvGT1) (21), along with the results of site-directed mutagenesis studies of these enzymes (22), afforded important information relative to the mechanism and specificity of PSPG-catalyzed reactions. In both of these enzymes, a histidine residue (His-22 in UGT71G1 and His-20 in VvGT1) that is highly conserved among PSPGs is proposed to act as a key catalytic residue that activates the hydroxy group of the glucosyl acceptor molecule to facilitate glucosidic linkage formation.…”

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confidence: 99%

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

Niimi

Saito

Homma

et al. 2007

Journal of Biological Chemistry

122

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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.

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Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

Cited by 113 publications

References 38 publications

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

Catalytic Key Amino Acids and UDP-Sugar Donor Specificity of a Plant Glucuronosyltransferase, UGT94B1: Molecular Modeling Substantiated by Site-Specific Mutagenesis and Biochemical Analyses

A single amino acid in the PSPG‐box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

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

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