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Open AccessArticle

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

1
Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
2
College of Sciences, Nanjing Agricultural University, Nanjing 210095, China
3
Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China
*
Authors to whom correspondence should be addressed.
Academic Editors: Jose M. Palomo and Cesar Mateo
Catalysts 2016, 6(12), 212; https://doi.org/10.3390/catal6120212
Received: 26 November 2016 / Revised: 11 December 2016 / Accepted: 12 December 2016 / Published: 17 December 2016
(This article belongs to the Special Issue Asymmetric and Selective Biocatalysis)
The control of cellular N-acetylmannosamine (ManNAc) levels has been postulated to be an effective way to modulate the decoration of cell surfaces with sialic acid. N-acetylglucosamine 2-epimerase catalyzes the interconversion of N-acetylglucosamine (GlcNAc) and ManNAc. Herein, we describe the cloning, expression, purification and biochemical characterization of an unstudied N-acetylglucosamine 2-epimerase from Pedobacter heparinus (PhGn2E). To further characterize the enzyme, several N-acylated glucosamine derivatives were chemically synthesized, and subsequently used to test the substrate specificity of PhGn2E. Furthermore, NMR studies of deuterium/hydrogen exchange at the anomeric hydroxy group and C-2 positions of the substrate in the reaction mixture confirmed for the first time the postulated epimerization reaction via ring-opening/enolate formation. Site-directed mutagenesis of key residues in the active site showed that Arg63 and Glu314 are directly involved in proton abstraction and re-incorporation onto the substrate. As all mechanistically relevant active site residues also occur in all mammalian isoforms, PhGn2E can serve as a model N-acetylglucosamine 2-epimerase for further elucidation of the active site mechanism in these enzymes. View Full-Text
Keywords: sialic acid metabolism; N-acetylglucosamine 2-epimerase; deprotonation/reprotonation mechanism; Neu5Ac analogues; synthesis of sialic acid analogues sialic acid metabolism; N-acetylglucosamine 2-epimerase; deprotonation/reprotonation mechanism; Neu5Ac analogues; synthesis of sialic acid analogues
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MDPI and ACS Style

Wang, S.-Y.; Laborda, P.; Lu, A.-M.; Duan, X.-C.; Ma, H.-Y.; Liu, L.; Voglmeir, J. N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism. Catalysts 2016, 6, 212.

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