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 12 2022
Accepted
21 2 2023
First published
21 2 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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