Analysis of substrate specificity of galactinol synthase from Brachypodium dystachion

MARTINEZ, MARÍA P.; MINEN, ROMINA I.; IGLESIAS, ALBERTO A.; FIGUEROA, CARLOS M.

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB y PABMB

Raffinose (Raf) is an α-1,6-galactosyl extension ofsucrose that plays a key role in the stabilization of membranes during seeddesiccation. Plants from the Cucurbitaceae family also use Raf for transportingcarbon from photosynthetic to heterotrophic tissues. Additionally, Rafaccumulates in tissues exposed to several abiotic stress conditions, such asheat, cold, salinity and drought. Considering the importance of Raf for plantphysiology and biochemistry, the number of studies dealing with enzymesinvolved in Raf biosynthesis are relatively scarce. To better understand thekinetic and regulatory properties of the enzymes involved in Raf metabolism, wecloned the genes encoding UDP-sugar pyrophosphorylase (USPPase, EC 2.7.7.64),galactinol synthase (EC 2.4.1.123) and Raf synthase (EC2.4.1.82) from Brachypodium distachyon, a model grassevolutionary related to several economically important species, including riceand wheat. USPPase catalyzes the production of activated sugars, mainly UDP-galactose(UDP-Gal), the natural substrate of galactinol synthase, which in turn producesgalactinol from UDP-Gal and myo-inositol.Afterward, Raf synthase transfers the Gal moiety from galactinol to a preformedsucrose molecule to produce Raf. The genes encoding USPPase, galactinolsynthase and Raf synthase from B. distachyonwere synthesized de novo and therecombinant proteins were expressed with an N-term His-tag in Escherichia coli cells and purified in asingle step by IMAC. The activity of USPPase with different hexoses-1P was asfollows: Gal‑1P ~ glucose-1P> glucuronic acid-1P > mannose-1P > glucosamine-1P. We found that galactinolsynthase uses UDP-glucose as an alternative substrate, although with a significantlylower catalytic efficiency (196 M-1 s-1) than with UDP-Gal(2.6 x 105 M-1 s-1). Our results suggest that galactinolsynthase might use other UDP-sugarsinvitro, thus leading to novel myo-inositolderivatives different from galactinol. These molecules could then be used byRaf synthase to produce original oligosaccharides with multiple applications inbiorefineries.