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

Abstract

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.

Synthesis of Sialic Acids, Their Derivatives, and Analogs by Using a Whole‐Cell Catalyst

“…In addition to its natural substrates GlcNAc ( 1 ) and ManNAc ( 10 ), AGE accepted a range of GlcNAc derivatives ( 2 – 8 ) with modifications at different positions of the sugar backbone, affording their corresponding epimerization products ( 18 – 24 ) in 9–35 % equilibrium yields. No product formation was confirmed for the AGE‐catalyzed reactions of all the non‐nitrogenous sugars tested ( 11 – 16 ), in agreement with the catalytic mechanism of AGE reported previously . In contrast to AGE, NanA accepted ManNAc and the non‐nitrogenous sugars, but not GlcNAc and its derivatives as substrates, consistent with previous studies .…”

“…Both reactions are reversible. The characteristics of substrates acceptable to both AGE and NanA have been well studied previously. Sixteen aldoses were examined here and the results are summarized in Table .…”

The crystal structure of theN-acetylglucosamine 2-epimerase fromNostocsp. KVJ10 reveals the true dimer

“…It shows that the ethylene glycol molecule in the active site of nAGE10 is in proximity to the O5, C5, C6 and O6 positions of mannose, as well as the corresponding positions of cellobiitol. This placement is consistent with the suggested role of Arg63 of the epimerase PhGn2E as the agent that is responsible for the protonation and deprotonation of ManNAc (Wang et al, 2016). Indeed, the corresponding position in AGE10 (Ar68) is only 2.9 Å from the oxygen of ethylene glycol that mimicks O5 of mannose (Fig.…”

“…Together with mutagenesis studies, this led to the proposal of a reaction mechanism involving two critical histidine residues as acid/base catalysts in the protonation/ deprotonation of carbon C2 of GlcNAc/ManNAc. The enzyme from Pedobacter heparinus DSM2366 (PhGn2E) was used in a hydrogen/deuterium-exchange experiment, which confirmed this type of mechanism for the epimerization, albeit with a glutamine/arginine pair as catalysts (Wang et al, 2016).…”

“…It seems that their role, at least in mammals, is to divert the metabolic flux away from sialic acid synthesis (Luchansky et al, 2003). The reaction follows a deprotonation/reprotonation mechanism involving two key residues, which had been thought to be histidines, but which have recently been identified as a glutamine and an arginine (Lee, Wu et al, 2007;Wang et al, 2016). AGE activity is greatly enhanced by the presence of nucleotides, in particular ATP, which serve as allosteric activators (Datta, 1970;Ghosh & Roseman, 1965;Tabata et al, 2002;Takahashi, Hori et al, 2001).…”