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

Muchut, Robertino J., Calloni, Rodrigo D., Herrera, Fernando E., Garay, Sergio A., Arias, Diego G., Iglesias, Alberto A., Guerrero, Sergio A.
Biochimie 2018 v.154 pp. 176-186
Escherichia coli, Euglena gracilis, carbohydrate metabolism, catalytic activity, cysteine, disulfide bonds, dithiothreitol, electrophoresis, flagellum, fluorescence microscopy, genes, glycosyltransferases, heterologous gene expression, hydrogen peroxide, molecular cloning, oxidation, polysaccharides, thioredoxins, uridine triphosphate
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 3350⁻¹. 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.