N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism
AbstractThe 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
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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.
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(12):212.Chicago/Turabian Style
Wang, Su-Yan; Laborda, Pedro; Lu, Ai-Min; Duan, Xu-Chu; Ma, Hong-Yu; Liu, Li; Voglmeir, Josef. 2016. "N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism." Catalysts 6, no. 12: 212.
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