Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase.

MUCHUT, ROBERTINO; CALLONI, RODRIGO; ARIAS, DIEGO G.; HERRERA, FERNANDO E.; GARAY, ALBERTO S.; IGLESIAS, ALBERTO A.; GUERRERO, SERGIO A.

BIOCHIMIE

ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER

Lugar: Paris; Año: 2018 vol. 154 p. 176 - 186

0300-9084

Many oligoand polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they aresynthesized by glycosyl transferases using UDP-glucose as a substrate. Herein,we report the molecular cloning of a gene putatively coding for a UDP-glucosepyrophosphorylase (EgrUDP-GlcPPase)in E. gracilis. After heterologousexpression of the gene in Escherichiacoli, the recombinant enzyme was characterized structural and functionally.Highly purified EgrUDP-GlcPPaseexhibited a monomeric structure, able to catalyze synthesis of UDP-glucose witha Vmax of 3,350 U.mg-1.Glucose‑1P and UTP were the preferred substrates, although the enzyme also used(with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidationby hydrogen peroxide inactivated the enzyme, an effect reversed by reductionwith dithiothreitol or thioredoxin. The redox process would involve sulfenicacid formation, since no pair of the 7 cysteine residues is close enough in the3D structure of the protein to form a disulfide bridge. Electrophoresis studiessuggest that, after oxidation, the enzyme arranges in many enzymaticallyinactive structural conformations; which were also detected in vivo. Finally, confocal fluorescencemicroscopy provided evidence for a cytosolic (mainly in the flagellum)localization of the enzyme.