info:eu-repo/semantics/article
Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase
Fecha
2018-11Registro en:
Muchut, Robertino José; Calloni, Rodrigo Daniel; Herrera, Fernando Enrique; Garay, Alberto Sergio; Arias, Diego Gustavo; et al.; Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase; Elsevier France-editions Scientifiques Medicales Elsevier; Biochimie; 154; 11-2018; 176-186
0300-9084
CONICET Digital
CONICET
Autor
Muchut, Robertino José
Calloni, Rodrigo Daniel
Herrera, Fernando Enrique
Garay, Alberto Sergio
Arias, Diego Gustavo
Iglesias, Alberto Daniel
Guerrero, Sergio Adrian
Resumen
Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 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. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.