کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1387755 1500835 2015 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Probing of the reaction pathway of human UDP-xylose synthase with site-directed mutagenesis
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آلی
پیش نمایش صفحه اول مقاله
Probing of the reaction pathway of human UDP-xylose synthase with site-directed mutagenesis
چکیده انگلیسی


• UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-glucuronic acid.
• UXS is similar to UDP-glucuronic acid 4-epimerase (UGAE).
• Active site of UXS was changed to resemble that of UGAE.
• Reaction pathway of UXS mutants was analyzed.
• Mutants produce UDP-4-keto-pentose intermediate besides UDP-xylose.

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 (Glu120 and Arg277) are replaced by Ser and Thr in the UGAE group. Additionally, Asn176, 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.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Carbohydrate Research - Volume 416, 30 October 2015, Pages 1–6
نویسندگان
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