دورية أكاديمية
Real-Time Monitoring of the Sialic acid Biosynthesis Pathway by NMR
| العنوان: | Real-Time Monitoring of the Sialic acid Biosynthesis Pathway by NMR |
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| المؤلفون: | Jacob L. Gorenflos López, Peter Schmieder, Kristin Kemnitz-Hassanin, Arif Celik, Christian Stieger, Dorothea Fiedler, Stephan Hinderlich, Christian P. R. Hackenberger |
| المصدر: | Chemical Science |
| سنة النشر: | 2023 |
| المجموعة: | Zenodo |
| مصطلحات موضوعية: | Sialic acid Biosythesis, Real-Time NMR, Assay Development |
| الوصف: | Sialic acids are part of the outermost component of the glycocalyx of all vertebrates; as such, they are fundamental markers in physiological and pathological processes. In this study, we introduce a real-time assay to monitor individual enzymatic steps of sialic acid biosynthesis, either with recombinant enzymes, in particular using UDP-N-acetylglucosamine 2-epimerase (GNE) or N-acetylmannosamine kinase (MNK), or in cytosolic rat liver extract. Using state-of-the-art NMR techniques, we are able to follow the characteristic signal of the N-acetyl methyl group, which displays different chemical shifts for the biosynthesis intermediates UDP-N-acetylglucosamine, N-acetylmannosamine (and its 6-phosphate) and N-acetylneuraminic acid (and its 9-phosphate). Pseudo 2- and 3-D NMR demonstrated that in rat liver cytosolic extract, the phosphorylation reaction of MNK is exclusive for N-acetylmannosamine generated by GNE. Thus, we speculate that phosphorylation of this sugar from other sources (e.g. external application to cells) or N-acetylmannosamine derivatives often applied in metabolic glycoengineering is not conducted by MNK but by a yet unknown sugar kinase. Competition experiments with the most prevalent neutral carbohydrates demonstrated that of these, only N-acetylglucosamine slowed N-acetylmannosamine phosphorylation kinetics, suggesting an N-acetylglucosamine-preferring kinase as the acting enzyme. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| العلاقة: | https://zenodo.org/record/7648851Test; https://doi.org/10.5281/zenodo.7648851Test; oai:zenodo.org:7648851 |
| DOI: | 10.5281/zenodo.7648851 |
| الإتاحة: | https://doi.org/10.5281/zenodo.7648851Test https://doi.org/10.5281/zenodo.7648850Test https://zenodo.org/record/7648851Test |
| حقوق: | info:eu-repo/semantics/openAccess ; https://creativecommons.org/licenses/by/4.0/legalcodeTest |
| رقم الانضمام: | edsbas.571DC6C2 |
| قاعدة البيانات: | BASE |
| DOI: | 10.5281/zenodo.7648851 |
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