Biochemical characterization of recombinant UDP-sugar pyrophosphorylase and galactinol synthase fromBrachypodium distachyon

Raffinose (Raf) protects plant cells during seed desiccation and under different abiotic stress conditions. The biosynthesis of Raf starts with the production of UDP-galactose by UDP-sugar pyrophosphorylase (USPPase) and continues with the synthesis of galactinol by galactinol synthase (GolSase). Galactinol is then used by Raf synthase to produce Raf. In this work, we report the biochemical characterization of USPPase ( Bdi USPPase) and GolSase 1 ( Bdi GolSase1) from Brachypodium distachyon . The catalytic efficiency of Bdi USPPase was similar with galactose 1-phosphate and glucose 1-phosphate, but 5-to 17-fold lower with other sugar 1-phosphates. The catalytic efficiency of Bdi GolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of Bdi GolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys 261 lies within the putative catalytic pocket. Bdi GolSase1 was inactivated by oxidation with diamide and H 2 O 2 . The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that Bdi GolSase1 is redox-regulated.