cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures

Nagashima, Shigeyuki; Inagaki, Rieko; Kubo, Akiko; Hirotani, Masao; Yoshikawa, Takafumi

doi:10.1007/s00425-003-1118-0

Cited by 42 publications

(27 citation statements)

References 22 publications

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“…It also shows high sequence similarities (60 -71% identity) to PSPG homologs of unknown biochemical function (Vigna angularis (adzuki beans) glucosyltransferase-1, -3, and 5; accession numbers BAB86919, BAB86921, and BAB86923, respectively). Notably, however, GmIF7GT shows only low sequence similarity (29% identity) to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase (GeIF7GT (UGT73F1); accession number BAC78438) (33), which displays a substrate specificity that is very similar to that of GmIF7GT. GmIF7GT is 32 and 25% identical in primary structure to PSPGs from M. truncatula (UGT71G1; accession number AAW56092) and V. vinifera (VvGT1; accession number AAB81682), respectively, the crystal structures of which were clarified recently (20,21).…”

Section: Cdna Cloning Heterologous Expression and Sequence Comparisonmentioning

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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Section: Cdna Cloning Heterologous Expression and Sequence Comparisonmentioning

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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“…2b), of the amino acid residue corresponding to Cys377 in CaUGT2 and the ability of these enzymes to glycosylate curcumin. As shown in Table 3, flavonoid-7-O-glucosyltransferase (F7GT) from Scutellaria baicalensis [28] with Cys368, betanidin 5-O-glucosyltransferase (B5GT) from Dorotheanthus bellidiformis [29] with Ser381, UGT73F1 from Glycyrrhiza echinata [30] with Ser370, and UGT73C6 from Arabidopsis thaliana [31] with Thr384 as amino acid residues corresponding to Cys377 in CaUGT2 displayed some enzymatic activity toward curcumin, whereas steroidal saponin synthase (SaGT4A) from Solanum aculeatissimum [32] with Ile380 was unable to catalyze the glucosylation of curcumin. Although the K m value for either curcumin or UDP-glucose fluctuated irrespective of the amino acid residues, k cat values are higher for the enzymes with Cys than for those with Ser or Thr at that position.…”

Section: Reciprocal Site-directed Mutagenesismentioning

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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“…The Arabidopsis Genome Initiative codes corresponding to Arabidopsis UGTs are given in Table II. The GenBank accession numbers of other plant GTs are: Allium cepa 73G1 and 73J1, AAP88406 and AAP88407, respectively (Kramer et al, 2003); Catharanthus roseus, BAD29722 (Kaminaga et al, 2004); Dorotheanthus bellidiformis, CAB56231 (Vogt et al, 1999); Gentiana triflora, BAC54092 (Fukuchi-Mizutani et al, 2003); Glycyrrhiza echinata, BAC78438 (Nagashima et al, 2004); tomato, CAA59450 (O'Donnell et al, 1998); Manihot esculenta, CAA54610 (Hughes and Hughes, 1994); Medicago truncatula, AAW56091 (Achnine et al, 2005); tobacco, TOGT1 and TOGT2, AAK28303 and AAK28304, respectively (Fraissinet-Tachet et al, 1998); Scutellaria baicalensis, BAA83484 (Hirotani et al, 2000); Solanum tuberosum, AAB48444 (Moehs et al, 1997); and Vigna angularis, BAB83692 (Xu et al, 2002).…”

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confidence: 99%

Pathogen-Responsive Expression of Glycosyltransferase Genes UGT73B3 and UGT73B5 Is Necessary for Resistance to Pseudomonas syringae pv tomato in Arabidopsis

2005

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The genome sequencing of Arabidopsis (Arabidopsis thaliana) has revealed that secondary metabolism plant glycosyltransferases (UGTs) are encoded by an unexpectedly large multigenic family of 120 members. Very little is known about their actual function in planta, in particular during plant pathogen interactions. Among them, members of the group D are of particular interest since they are related to UGTs involved in stress-inducible responses in other plant species. We provide here a detailed analysis of the expression profiles of this group of Arabidopsis UGTs following infection with Pseudomonas syringae pv tomato or after treatment with salicylic acid, methyljasmonate, and hydrogen peroxide. Members of the group D displayed distinct induction profiles, indicating potential roles in stress or defense responses notably for UGT73B3 and UGT73B5. Analysis of UGT expression in Arabidopsis defense-signaling mutants further revealed that their induction is methyljasmonate independent, but partially salicylic acid dependent. T-DNA tagged mutants (ugt73b3 and ugt73b5) exhibited decreased resistance to P. syringae pv tomato-AvrRpm1, indicating that expression of the corresponding UGT genes is necessary during the hypersensitive response. These results emphasize the importance of plant secondary metabolite UGTs in plant-pathogen interactions and provide foundation for future understanding of the exact role of UGTs during the hypersensitive response.

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cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures

Cited by 42 publications

References 22 publications

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

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

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

Pathogen-Responsive Expression of Glycosyltransferase Genes UGT73B3 and UGT73B5 Is Necessary for Resistance to Pseudomonas syringae pv tomato in Arabidopsis

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