Kinetic and structural characterization of galactinol synthase from Brchypodium dystachion

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 is used for carbon export from source to sink tissues in manyplants. Raf plays an important role in the stabilization of membranes during seeddesiccation, being accumulated under certain abiotic stress conditions, includingheat, cold, salinity and drought. The first committed step in the pathway of Rafbiosynthesis is the reaction catalyzed by galactinol synthase (EC 2.4.1.123), amember of the glycosyltransferase family 8, which produces galactinol fromUDP-galactose (UDP-Gal) and myo-inositol.Then, raffinose synthase (EC 2.4.1.82) transfers the galactosyl moiety from galactinolto sucrose, thus producing Raf. Several works have shown the relationship of galactinolsynthase transcripts with galactinol and Raf levels in response to varioustypes of stressful conditions. However, little is known about the structural,kinetic and regulatory properties of this enzyme. The present work focuses ongalactinol synthase from Brachypodium distachyon,a model grass closely related to economically important crops, such as rice andwheat. The gene coding for this enzyme was synthesized de novo and the recombinant enzyme was expressed fused to an N-terminalHis-tag in Escherichia coli cells andpurified by IMAC. The activity of B.distachyon galactinolsynthase was 2-fold higher in presence of Mn2+ than with Mg2+,the enzyme exhibiting a 10-fold higher affinity for the former. Enzyme activityin the physiological direction of the reaction (synthesis of galactinol) wasoptimal at pH values from 7 to 9 and 40 °C. Under standard conditions, the S0.5 for UDP-Gal and myo-inositol were 0.08 and 2.9 mM,respectively. Interestingly, the recombinant enzyme was inactivated byoxidation with diamide and the activity was recovered by reduction with DTT,suggesting the existence of a redox regulatory mechanism. A structuralmodel of the enzyme was obtained by protein threading (fold recognition), whichwas used to determine the residues putativelyinvolved in substrate and metal binding. Overall, our work lays the ground tobetter understand the synthesis of Raf in plants.