Simultaneous one-pot multienzymatic synthesis and purification of Uridine-5′-Diphosphate-α-D-glucuronide and Uridine-5′-Diphosphate-α-d-xylose

IF 2.4 3区化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Carbohydrate Research Pub Date : 2025-05-13 DOI:10.1016/j.carres.2025.109516

Arnaud Pâris, Tiffany Rundstadler , Pierre Lafite

引用次数: 0

Abstract

Glucuronosyltransferases and xylosyltransferases are glycosyltransferases (GT) that are involved in a wide range of biological processes, including xenobiotic metabolism or proteoglycan biosynthesis and require as substrates activated sugar donors, respectively UDP-α-D-glucuronide (UDP-GlcA) and UDP-α-d-xylose (UDP-Xyl). As both nucleotide sugars are hardly available, we have developed a one-pot multienzymatic synthesis to produce sequentially UDP-GlcA and UDP-Xyl. This methodology involves the production of UDP-GlcA for glucuronic acid, coupling glucuronokinase and UDP-sugar pyrophosphorylase activities, followed by UDP-GlcA decarboxylation by UDP-Xylose Synthase. Each of these UDP-sugars can then be simultaneously purified and isolated from the unique reaction mixture, providing an efficient production methodology of these rare UDP-sugars for subsequent enzymatic studies of corresponding GT.

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尿苷-5′-二磷酸-α-d-葡萄糖醛酸和尿苷-5′-二磷酸-α-d-木糖的单锅多酶同时合成纯化

葡萄糖醛基转移酶和木糖基转移酶是参与多种生物过程的糖基转移酶（GT），包括异种生物代谢或蛋白聚糖生物合成，并需要作为底物的活化糖供体，分别是UDP-α-d-葡萄糖醛酸（UDP- glca）和UDP-α-d-木糖（UDP- xyl）。由于这两种核苷酸糖都很难获得，我们开发了一种一锅多酶合成方法，依次生产UDP-GlcA和UDP-Xyl。该方法包括生产葡萄糖醛酸的UDP-GlcA，偶联葡萄糖醛酸激酶和udp -糖焦磷酸化酶的活性，然后由udp -木糖合酶对UDP-GlcA进行脱羧。然后可以同时从独特的反应混合物中纯化和分离这些udp -糖，为后续相应GT的酶促研究提供这些罕见的udp -糖的有效生产方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Carbohydrate Research 化学-生化与分子生物学

CiteScore

5.00

自引率

3.20%

发文量

183

审稿时长

3.6 weeks

期刊介绍： Carbohydrate Research publishes reports of original research in the following areas of carbohydrate science: action of enzymes, analytical chemistry, biochemistry (biosynthesis, degradation, structural and functional biochemistry, conformation, molecular recognition, enzyme mechanisms, carbohydrate-processing enzymes, including glycosidases and glycosyltransferases), chemical synthesis, isolation of natural products, physicochemical studies, reactions and their mechanisms, the study of structures and stereochemistry, and technological aspects. Papers on polysaccharides should have a "molecular" component; that is a paper on new or modified polysaccharides should include structural information and characterization in addition to the usual studies of rheological properties and the like. A paper on a new, naturally occurring polysaccharide should include structural information, defining monosaccharide components and linkage sequence. Papers devoted wholly or partly to X-ray crystallographic studies, or to computational aspects (molecular mechanics or molecular orbital calculations, simulations via molecular dynamics), will be considered if they meet certain criteria. For computational papers the requirements are that the methods used be specified in sufficient detail to permit replication of the results, and that the conclusions be shown to have relevance to experimental observations - the authors'' own data or data from the literature. Specific directions for the presentation of X-ray data are given below under Results and "discussion".