Issue 13, 2023

Real-time monitoring of the sialic acid biosynthesis pathway by NMR

Abstract

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.

Graphical abstract: Real-time monitoring of the sialic acid biosynthesis pathway by NMR

Supplementary files

Article information

Article type
Edge Article
Submitted
20 דצמ 2022
Accepted
21 פבר 2023
First published
21 פבר 2023
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2023,14, 3482-3492

Real-time monitoring of the sialic acid biosynthesis pathway by NMR

J. L. Gorenflos López, P. Schmieder, K. Kemnitz-Hassanin, H. C. Asikoglu, A. Celik, C. E. Stieger, D. Fiedler, S. Hinderlich and C. P. R. Hackenberger, Chem. Sci., 2023, 14, 3482 DOI: 10.1039/D2SC06986E

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