The State Key Laboratory of Microbial Technology and School of Life Sciences, Jinan, Shandong 250100, China.
Bioorg Med Chem Lett. 2013 Jul 1;23(13):3764-8. doi: 10.1016/j.bmcl.2013.04.090. Epub 2013 May 9.
Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.
核苷酸糖是单糖的活化形式，也是所有生物碳水化合物代谢的关键中间产物。结构多样的核苷酸糖的可用性对于糖基转移酶的表征尤为重要。鉴于核苷酸糖，尤其是其有用的非天然衍生物的制备方法有限，我们在此引入了一种从单糖一步合成核苷酸糖的高效三步酶催化体系。在这项研究中，我们使用来自拟南芥（AtUSP）的混杂型 UDP-糖焦磷酸化酶（USP）与肺炎链球菌 TIGR4 的半乳糖激酶（SpGalK）和无机焦磷酸酶（PPase）有效地合成了四种 UDP-糖。与来自肺炎链球菌 TIGR4 的 UDP-葡萄糖焦磷酸化酶（SpGalU）相比，AtUSP 对 Gal-1-P 的 C4 衍生物具有更好的耐受性。此外，我们还系统地研究了 AtUSP 的核苷酸底物特异性和动力学参数。AtUSP 对 UTP、dUTP 和 dTTP 表现出相当的活性，其产率分别为 87%、85%和 84%。这些结果为更好地理解 AtUSP 的底物特异性与结构特征之间的关系提供了丰富的信息。
