Eixelsberger Thomas, Weber Hansjörg, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, NAWI Graz, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Institute of Organic Chemistry, NAWI Graz, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria.
Carbohydr Res. 2015 Oct 30;416:1-6. doi: 10.1016/j.carres.2015.08.006. Epub 2015 Aug 17.
Uridine 5'-diphosphate (UDP)-xylose (UDP-Xyl) synthase (UXS) catalyzes the oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcUA) to UDP-Xyl. The closely related UDP-glucuronic acid 4-epimerase (UGAE) interconverts UDP-GlcUA and UDP-galacturonic acid (UDP-GalUA) in a highly similar manner via the intermediate UDP-xylo-hexopyranos-4-uluronic acid (UDP-4-keto-GlcUA). Unlike UXS, however, UGAE prevents the decarboxylation. Human UXS (hUXS) and UGAE from Arabidopsis thaliana exhibit high structural similarity in the active site, but two catalytically important residues in hUXS (Glu(120) and Arg(277)) are replaced by Ser and Thr in the UGAE group. Additionally, Asn(176), which participates in substrate binding, is changed to Thr. We therefore analyzed single, double and triple mutants of hUXS carrying these substitutions to evaluate their significance for product specificity. All mutants showed considerably lower activities than wild-type hUXS (>1000-fold reduction). NMR spectroscopic analysis of the reaction products showed that UDP-β-L-threo-pentopyranos-4-ulose (UDP-4-keto-Xyl), UDP-Xyl or both, but no UDP-GalUA or UDP-4-keto-GlcUA were formed. Correlation of product characteristics, such as deuterium incorporation, with the amino acid replacements gave insights into structure-function relationships in UXS, suggesting that interaction between active site and overall enzyme structure rather than distinct conserved residues are decisive for product formation.
尿苷5'-二磷酸（UDP）-木糖（UDP-Xyl）合酶（UXS）催化UDP-葡萄糖醛酸（UDP-GlcUA）氧化脱羧生成UDP-Xyl。密切相关的UDP-葡萄糖醛酸4-表异构酶（UGAE）以高度相似的方式通过中间产物UDP-木糖己吡喃糖-4-酮糖醛酸（UDP-4-酮基-GlcUA）使UDP-GlcUA和UDP-半乳糖醛酸（UDP-GalUA）相互转化。然而，与UXS不同的是，UGAE可防止脱羧反应。人UXS（hUXS）和拟南芥的UGAE在活性位点具有高度的结构相似性，但hUXS中两个具有催化重要性的残基（Glu(120)和Arg(277)）在UGAE组中被Ser和Thr取代。此外，参与底物结合的Asn(176)变为Thr。因此，我们分析了携带这些替代的hUXS的单突变体、双突变体和三突变体，以评估它们对产物特异性的重要性。所有突变体的活性均比野生型hUXS低得多（降低超过1000倍）。对反应产物的核磁共振光谱分析表明，形成了UDP-β-L-苏式戊吡喃糖-4-酮糖（UDP-4-酮基-Xyl）、UDP-Xyl或两者，但未形成UDP-GalUA或UDP-4-酮基-GlcUA。产物特征（如氘掺入）与氨基酸替代之间的相关性为UXS的结构-功能关系提供了见解，表明活性位点与整个酶结构之间的相互作用而非特定的保守残基对产物形成起决定性作用。
