A multifunctional galactinol synthase catalyzes the synthesis of fagopyritol A1 and fagopyritol B1 in buckwheat seed

Fagopyritols are galactosyl cyclitols in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) seeds with structural similarities to a putative insulin mediator deficient in non-insulin dependent diabetes mellitus and polycystic ovary syndrome. Based on the assumption that the multifunctional enzyme galactinol synthase (GolS, UDP-galactose:myo-inositol galactosyltransferase, EC 2.4.1.123) may have homology to the enzyme responsible for the synthesis of fagopyritols, we isolated two full-length and one partial cDNA clones encoding GolS homologues from buckwheat seeds. We designated the genes corresponding to these three cDNAs as Fagopyrum esculentum GolS (FeGolS)1, FeGolS-2 and FeGolS-3. The full-length FeGolS-1 and FeGolS-2 cDNAs are 1269 bp and 1326 bp in length and encode polypeptides of 38.3 kDa and 40.7 kDa, respectively. According to the deduced amino acid sequences, FeGolS-1 and FeGolS-2 share a high level of sequence similarity with GolSs in other species. FeGolS-2 and FeGolS-3 contain an insertion of 17 or 18 amino acid residues near the carboxyl terminus, respectively, which is absent in FeGolS-1 and other GolSs. Both bacterially expressed recombinant FeGolS-1 and FeGolS-2 proteins exhibited GolS activities when assayed in the presence of UDP-galactose and myo-inositol. In the presence of UDP-galactose and D-chiro-inositol, FeGolS-1 catalyzed the synthesis of fagopyritol B1 whereas FeGolS-2 catalyzed the synthesis of both fagopyritol A1 and fagopyritol B I in a 1:4 mole ratio. These results demonstrated that multifunctional GolS homologues in buckwheat seeds confer fagopyritol synthase activities and that the specificity for fagopyritol A1 synthesis may be mediated by a unique class of GolS homologues. (C) 2004 Elsevier Ireland Ltd. All rights reserved.