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Catalysts 2016, 6(12), 212; doi:10.3390/catal6120212

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
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)
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Abstract

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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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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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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