Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation

Kubo, Akiko; Arai, Yuka; Nagashima, Shigeyuki; Yoshikawa, Takafumi

doi:10.1016/j.abb.2004.06.021

Cited by 150 publications

(131 citation statements)

References 37 publications

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“…The amino acids Gln-375, Asp-374, and Thr-141 have been proposed to interact with hydroxyl groups at the C-2 and C-3, C-3, and C-4, and C-6 positions of the glucose moiety of UDPglucose, respectively, but Gln-375 and Thr-141 are not conserved in the Arabidopsis 3GlyTs (see Supplemental Figure 3 online). His residues corresponding to Gln-375 are conserved among the galactosyltransferases (Kubo et al, 2004). A His residue was also found as the corresponding residue in UGT78D3.…”

Section: A Flavonol Glycosyltransferase:flavonol 3-o-arabinosyltransfmentioning

confidence: 84%

Comprehensive Flavonol Profiling and Transcriptome Coexpression Analysis Leading to Decoding Gene–Metabolite Correlations inArabidopsis

et al. 2008

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To complete the metabolic map for an entire class of compounds, it is essential to identify gene-metabolite correlations of a metabolic pathway. We used liquid chromatography-mass spectrometry (LC-MS) to identify the flavonoids produced by Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-Oarabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase. Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDPrhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the identification of a gene function and a more complete understanding of a plant metabolic network.

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Section: A Flavonol Glycosyltransferase:flavonol 3-o-arabinosyltransfmentioning

confidence: 84%

Comprehensive Flavonol Profiling and Transcriptome Coexpression Analysis Leading to Decoding Gene–Metabolite Correlations inArabidopsis

et al. 2008

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“…By molecular modeling, the effect of the Q373H substitution could be explained in terms of a slight shift in the binding of the sugar donor molecule in the enzyme upon this substitution (see Supplemental Figure 4 online). It is important to note, however, that Vv GT1 also displayed low GalT activity, which was ;8% of its GlcT activity (in terms of k cat /K m value); and unlike the cases of Vv GT6 and other enzymes (Kubo et al, 2004), a Q375H substitution in Vv GT1 does not improve its GalT activity (Offen et al, 2006). Thus, the effect of the Gln/His substitution at that position on sugar donor specificity differs among UGTs.…”

Section: Specificity Determinants Of Vv Gt6 For Sugar Donorsmentioning

confidence: 93%

“…Judging from the occurrence of Gln at the C-terminal position of the PSPG box sequence (Mato et al, 1998;Miller et al, 1999;Kubo et al, 2004), the bifunctional Vv GT6 could be considered a variant of GlcT with an enhanced GalT activity. Replacement of Gln-373 with His consistently resulted in loss of Vv GT6 activity toward UDP-Glc, without substantial loss of GalT activity, giving rise to a monofunctional GalT (Table 5).…”

Section: Specificity Determinants Of Vv Gt6 For Sugar Donorsmentioning

confidence: 99%

“…We explored characteristics that are unique to flavonoid GlcTs and those unique to flavonoid GalTs by means of sequence comparison as well as molecular modeling of a variety of flavonoid GlcTs and GalTs. Previous site-directed mutagenesis studies using a flavonoid GlcT and a flavonoid GalT showed that an amino acid residue located at the C-terminal position of the PSPG box sequence (Gln and His, respectively; see Figure 4C) critically determines the sugar donor specificity of these enzymes: Gln at that position is important for the maximal catalytic efficiency of GlcT activity, whereas His is required for GalT activity (Kubo et al, 2004); Vv GT6 has a Gln reside (Gln-373) at this position. Moreover, position 19 of Vv GT6 is uniquely occupied by a Pro residue, whereas the corresponding position in known flavonoid GlcTs and GalTs is occupied by Thr or Ser ( Figure 4C).…”

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confidence: 99%

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

et al. 2010

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We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-Oglucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities. INTRODUCTIONGrapevine (Vitis vinifera) ( Figure 1A) has been one of the most important fruit crops from the beginning of human civilization (McGovern et al., 1997). Dietary intake of grapevine-based products results in diverse biological effects that are beneficial to human health, in part due to polyphenols, such as flavonoids (e.g., flavonols, proanthocyanidins, and anthocyanins) (Renaud and de Lorgeril, 1992;Corder et al., 2001Corder et al., , 2006Baur et al., 2006). Flavonols, such as kaempferol, quercetin, and isorhamnetin, are an important class of bioactive flavonoids, which are most abundant as their 3-O-glycosides (i.e., glucoside, glucuronoside, galactoside, and rutinoside) in the berries, seeds, and leaves of grapevine (Hmamouchi et al., 1996;Castillo-Munoz et al., 2009).In this plant, biosynthesis of flavonol glycosides primarily serves to protect the plant from excess UV light (Price et al., 1995;Matus et al., 2009). Flavonol aglycons are biosynthesized from dihydroflavonols by the action of flavonol synthase (Vv FLS), a 2-oxoglutarate-dependent dioxygenase, which is temporally regulated by an R2R3-Myb transcription factor, Vv MYBF1 (Fujita et al., 2006;Czemmel et al., 2009). Subsequently, glycosylation of flavonols enhances their water solubility, such that high concentrations of flavonols can accumulate in plant cells.From a food chemistry perspective, flavonol glycosides define the color of wine grapes and products by acting as copigments (Yoshitama et al., 1992;Boulto...

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“…Plant UGTs capable of transferring sugars to a wide range of small molecules belong to Family 1, and are defined by the presence of a 44 amino acid C-terminal signature motif designated as the PSPG (plant secondary product glycosyltransferases)-box [11]. Biochemical and molecular characterization of plant Family 1 UGTs has shown that they have relatively broad substrate specificity but exert regioselectivity towards sugar acceptors [12,13].…”

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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Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation

Cited by 150 publications

References 37 publications

Comprehensive Flavonol Profiling and Transcriptome Coexpression Analysis Leading to Decoding Gene–Metabolite Correlations inArabidopsis

Comprehensive Flavonol Profiling and Transcriptome Coexpression Analysis Leading to Decoding Gene–Metabolite Correlations inArabidopsis

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

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

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