Biochemical characterization of recombinant UDP-sugar pyrophosphorylase and galactinol synthase from Brachypodium...

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 (BdiUSPPase) and GolSase 1 (BdiGolSase1) from Brachypodium distachyon. The catalytic efficiency of BdiUSPPase 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 BdiGolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of BdiGolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys²⁶¹ lies within the putative catalytic pocket. BdiGolSase1 was inactivated by oxidation with diamide and H₂O₂. The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that BdiGolSase1 is redox-regulated.