Publications

• Purification, molecular cloning and functional characterization of flavonoid C-glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon.

  Nagatomo Y., Usui S., Ito T., Kato A., Shimosaka M., Taguchi G.

  C-Glycosides are characterized by their C-C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C-Glycosides are remarkably stable, as their C-C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accu ... >> More

  C-Glycosides are characterized by their C-C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C-Glycosides are remarkably stable, as their C-C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C-glycosylflavonoids; however, the genes encoding for enzymes that catalyze C-glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C-glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C-glucosylation of 2-hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C-glucosylation activity towards 2-hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2',4',6'-trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C-glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases. << Less

  Plant J. 80:437-448(2014) [PubMed] [EuropePMC]

• The C-glycosylation of flavonoids in cereals.

  Brazier-Hicks M., Evans K.M., Gershater M.C., Puschmann H., Steel P.G., Edwards R.

  Flavonoids normally accumulate in plants as O-glycosylated derivatives, but several species, including major cereal crops, predominantly synthesize flavone-C-glycosides, which are stable to hydrolysis and are biologically active both in planta and as dietary components. An enzyme (OsCGT) catalyzin ... >> More

  Flavonoids normally accumulate in plants as O-glycosylated derivatives, but several species, including major cereal crops, predominantly synthesize flavone-C-glycosides, which are stable to hydrolysis and are biologically active both in planta and as dietary components. An enzyme (OsCGT) catalyzing the UDP-glucose-dependent C-glucosylation of 2-hydroxyflavanone precursors of flavonoids has been identified and cloned from rice (Oryza sativa ssp. indica), with a similar protein characterized in wheat (Triticum aestivum L.). OsCGT is a 49-kDa family 1 glycosyltransferase related to known O-glucosyltransferases. The recombinant enzyme C-glucosylated 2-hydroxyflavanones but had negligible O-glucosyltransferase activity with flavonoid acceptors. Enzyme chemistry studies suggested that OsCGT preferentially C-glucosylated the dibenzoylmethane tautomers formed in equilibrium with 2-hydroxyflavanones. The resulting 2-hydroxyflavanone-C-glucosides were unstable and spontaneously dehydrated in vitro to yield a mixture of 6C- and 8C-glucosyl derivatives of the respective flavones. In contrast, in planta, only the respective 6C-glucosides accumulated. Consistent with this selectivity in glycosylation product, a dehydratase activity that preferentially converted 2-hydroxyflavanone-C-glucosides to the corresponding flavone-6C-glucosides was identified in both rice and wheat. Our results demonstrate that cereal crops synthesize C-glucosylated flavones through the concerted action of a CGT and dehydratase acting on activated 2-hydroxyflavanones, as an alternative means of generating flavonoid metabolites. << Less

  J. Biol. Chem. 284:17926-17934(2009) [PubMed] [EuropePMC]

• Identification and functional analysis of 2-hydroxyflavanone C-glucosyltransferase in soybean (Glycine max).

  Hirade Y., Kotoku N., Terasaka K., Saijo-Hamano Y., Fukumoto A., Mizukami H.

  C-Glucosyltransferase is an enzyme that mediates carbon-carbon bond formation to generate C-glucoside metabolites. Although it has been identified in several plant species, the catalytic amino acid residues required for C-glucosylation activity remain obscure. Here, we identified a 2-hydroxyflavan ... >> More

  C-Glucosyltransferase is an enzyme that mediates carbon-carbon bond formation to generate C-glucoside metabolites. Although it has been identified in several plant species, the catalytic amino acid residues required for C-glucosylation activity remain obscure. Here, we identified a 2-hydroxyflavanone C-glucosyltransferase (UGT708D1) in soybean. We found that three residues, His20, Asp85, and Arg292, of UGT708D1 were located at the predicted active site and evolutionarily conserved. The substitution of Asp85 or Arg292 with alanine destroyed C-glucosyltransferase activity, whereas the substitution of His20 with alanine abolished C-glucosyltransferase activity but enabled O-glucosyltransferase activity. The catalytic mechanism is discussed on the basis of the findings. << Less

  FEBS Lett. 589:1778-1786(2015) [PubMed] [EuropePMC]

• C-Glycosyltransferases catalyzing the formation of di-C-glucosyl flavonoids in citrus plants.

  Ito T., Fujimoto S., Suito F., Shimosaka M., Taguchi G.

  Citrus plants accumulate many kinds of flavonoids, including di-C-glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di-C-glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C-glycosylt ... >> More

  Citrus plants accumulate many kinds of flavonoids, including di-C-glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di-C-glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C-glycosyltransferases (CGTs) FcCGT (UGT708G1) and CuCGT (UGT708G2) as the primary enzymes involved in the biosynthesis of di-C-glucosyl flavonoids in the citrus plants kumquat (Fortunella crassifolia) and satsuma mandarin (Citrus unshiu), respectively. The amino acid sequences of these CGTs were 98% identical, indicating that CGT genes are highly conserved in the citrus family. The recombinant enzymes FcCGT and CuCGT utilized 2-hydroxyflavanones, dihydrochalcone, and their mono-C-glucosides as sugar acceptors and produced corresponding di-C-glucosides. The K_m and k_cat values of FcCGT toward phloretin were <0.5 μm and 12.0 sec^-1 , and those toward nothofagin (3'-C-glucosylphloretin) were 14.4 μm and 5.3 sec^-1 , respectively; these values are comparable with those of other glycosyltransferases reported to date. Transcripts of both CGT genes were found to concentrate in various plant organs, and particularly in leaves. Our results suggest that di-C-glucosyl flavonoid biosynthesis proceeds via a single enzyme using either 2-hydroxyflavanones or phloretin as a substrate in citrus plants. In addition, Escherichia coli cells expressing CGT genes were found to be capable of producing di-C-glucosyl flavonoids, which is promising for commercial production of these valuable compounds. << Less

  Plant J. 91:187-198(2017) [PubMed] [EuropePMC]