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 V_max 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.

许多寡糖和多糖（包括副mylon）在Euglena gracilis的生命周期中至关重要，它们是通过糖基转移酶以UDP-葡萄糖为底物合成的。在本文中，我们报道了推定的编码E. gracilis中的UDP-葡萄糖焦磷酸化酶（Egr UDP-GlcPPase）的基因的分子克隆。在大肠杆菌中异源表达该基因后，对重组酶进行了结构和功能表征。高度纯化的Egr UDP-GlcPPase显示出单体结构，能够催化UDP-葡萄糖的合成与V_最大3350 U.mg的^-1。葡萄糖-1P和UTP是优选的底物，尽管该酶也使用（催化效率较低）TTP，半乳糖-1P和甘露糖-1P。过氧化氢的氧化使酶失活，二硫苏糖醇或硫氧还蛋白的还原作用被逆转。氧化还原过程将涉及亚硫酸的形成，因为7个半胱氨酸残基中没有一对在蛋白质的3D结构中足够接近以形成二硫键。电泳研究表明，氧化后，该酶排列成许多无酶活性的结构构象。这在体内也被检测到。最后，共聚焦荧光显微镜检查为酶的胞质定位（主要在鞭毛中）提供了证据。