Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
J Biol Chem. 2011 Jun 17;286(24):21511-23. doi: 10.1074/jbc.M111.238774. Epub 2011 Apr 20.
The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.
植物 VTC2 基因编码 GDP-L-半乳糖磷酸化酶，这是植物维生素 C 生物合成中的限速酶。在哺乳动物中存在与 VTC2 明显同源的基因，这些动物通过独特的代谢途径产生维生素 C，而在线虫蠕虫秀丽隐杆线虫中尚未证明维生素 C 的生物合成。我们现在已经表达了人类和蠕虫 VTC2 同源基因（分别为 C15orf58 和 C10F3.4）的 cDNA，并发现纯化的蛋白质也显示 GDP-己糖磷酸化酶活性。然而，与植物酶相反，这些酶主要催化的反应是 GDP-D-葡萄糖的磷酸解为 GDP 和 D-葡萄糖 1-磷酸。我们在蠕虫和小鼠组织提取物中检测到具有类似底物特异性的活性。GDP-D-葡萄糖磷酸化酶的最高表达发生在神经系统和雄性生殖系统中。发现线虫 C10F3.4 缺失菌株完全缺乏 GDP-D-葡萄糖磷酸化酶活性；在用针对人类 C15orf58 基因的 siRNA 转染的人 HEK293T 细胞中，也发现这种活性降低。这些观察结果证实了对线虫 C10F3.4 和人类 C15orf58 基因表达产物的鉴定，它们是这些生物的 GDP-D-葡萄糖磷酸化酶。重要的是，我们发现 C10F3.4 突变体蠕虫中 GDP-D-葡萄糖的积累，表明 GDP-D-葡萄糖磷酸化酶可能作用于去除 GDP-D-葡萄糖，该酶先前被证明缺乏其生理 D-葡萄糖 1-磷酸底物的特异性。我们提出，这种去除可能防止错误掺入葡萄糖残基到糖缀合物中。
