Combining an Enhanced Polyphosphate Kinase-Driven UDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases for Efficient In Vitro Synthesis of Nucleoside Disaccharides,Journal of Agricultural and Food Chemistry

当前位置： X-MOL 学术 › J. Agric. Food Chem. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Combining an Enhanced Polyphosphate Kinase-Driven UDP-Glucose Regeneration System with the Screening of Key Glycosyltransferases for Efficient In Vitro Synthesis of Nucleoside Disaccharides
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-09-09 , DOI: 10.1021/acs.jafc.4c05329
Chuanqi Sun ₁ , Miaozi Lou ₁ , Zonglin Li ₁ , Feiyan Cheng ₁ , Zhimin Li _{1,

2}

Affiliation

Nucleoside disaccharides are essential glycosides that naturally occur in specific living organisms. This study developed an enhanced UDP-glucose regeneration system to facilitate the in vitro multienzyme synthesis of nucleoside disaccharides by integrating it with nucleoside-specific glycosyltransferases. The system utilizes maltodextrin and polyphosphate as cost-effective substrates for UDP-glucose supply, catalyzed by α-glucan phosphorylase (αGP) and UDP-glucose pyrophosphorylase (UGP). To address the low activity of known polyphosphate kinases (PPKs) in the UDP phosphorylation reaction, a sequence-driven screening identified RhPPK with high activity against UDP (>1000 U/mg). Computational design further led to the creation of a double mutant with a 2566-fold increase in thermostability at 50 °C. The enhanced UDP-glucose regeneration system increased the production rate of nucleoside disaccharide synthesis by 25-fold. In addition, our UDP-glucose regeneration system is expected to be applied to other glycosyl transfer reactions.

中文翻译：

将增强型多磷酸激酶驱动的 UDP-葡萄糖再生系统与关键糖基转移酶的筛选相结合，实现核苷二糖的高效体外合成

核苷二糖是天然存在于特定生物体中的必需糖苷。本研究开发了一种增强的UDP-葡萄糖再生系统，通过将其与核苷特异性糖基转移酶整合，促进核苷二糖的体外多酶合成。该系统利用麦芽糖糊精和多磷酸盐作为 UDP-葡萄糖供应的经济有效的底物，由 α-葡聚糖磷酸化酶 (αGP) 和 UDP-葡萄糖焦磷酸化酶 (UGP) 催化。为了解决 UDP 磷酸化反应中已知多磷酸激酶 (PPK) 的低活性问题，序列驱动的筛选鉴定出具有高 UDP 活性 (>1000 U/mg) 的 RhPPK。计算设计进一步导致了双突变体的创建，其在 50 °C 下的热稳定性提高了 2566 倍。增强的UDP-葡萄糖再生系统将核苷二糖合成的生产率提高了25倍。此外，我们的UDP-葡萄糖再生系统有望应用于其他糖基转移反应。

更新日期：2024-09-09

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南