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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 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.