| Autor: |
Su-Yan Wang, Pedro Laborda, Ai-Min Lu, Xu-Chu Duan, Hong-Yu Ma, Li Liu, Josef Voglmeir |
| Jazyk: |
angličtina |
| Rok vydání: |
2016 |
| Předmět: |
|
| Zdroj: |
Catalysts, Vol 6, Iss 12, p 212 (2016) |
| Druh dokumentu: |
article |
| ISSN: |
2073-4344 |
| DOI: |
10.3390/catal6120212 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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