Efficient Glycosynthase Mutant Derived from Mucor hiemalis Endo-β-N-acetylglucosaminidase Capable of Transferring Oligosaccharide from Both Sugar Oxazoline and Natural N-Glycan

Midori Umekawa,Cishan Li,Takayuki Higashiyama,Wei Huang,Hisashi Ashida,Kenji Yamamoto,Lai-Xi Wang

doi:10.1074/jbc.m109.059832

Midori Umekawa, Cishan Li + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.059832

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Abstract

Endo-M, an endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is a family 85 glycoside hydrolase. This enzyme is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates. In this study, we performed mutational and kinetic studies focusing on a key catalytic asparagine 175 of Endo-M. We have shown that most of the Asn-175 mutants had significantly diminished hydrolysis activity but acted as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation. Our results confirm the critical role of this asparagine residue in promoting the formation of an oxazolinium ion intermediate in the first step of the substrate-assisted catalysis. Interestingly, the N175Q mutant was found to possess dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with "natural" N-glycan as well. These results also implicated the significance of amide side chain in the asparagine 175 of Endo-M for promoting oxazoline transglycosylation in the second step of the catalysis. The highly efficient syntheses of glycopeptides/glycoproteins by N175Q combined with synthetic sugar oxazolines or natural N-glycan substrates were exemplified. In addition, we also identified several previously unknown residues that seem to play a role in the catalysis of Endo-M.

Highlights

JANUARY 1, 2010 VOLUME 285 NUMBER 1 between the N,NЈ-diacetylchitobiose core of N-glycans
Site-directed Saturation Mutagenesis at Asn-175—We previously found that the N175A mutant of Endo-M behaves as a “glycosynthase”; it can use the activated sugar oxazoline as a donor substrate for transglycosylation, but it lacks the activity to hydrolyze the product formed [7]
We have previously found that the N175A mutant of Endo-M exhibited glycosynthase-like activity using sugar oxazoline as a donor substrate, suggesting the critical participation of Asn-175 residue in the formation of an oxazoline intermediate, the first step of substrate-assisted catalysis

Summary

Introduction

JANUARY 1, 2010 VOLUME 285 NUMBER 1 between the N,NЈ-diacetylchitobiose core of N-glycans. Structural and mechanistic studies with some GH18 ENGases and GH18 chitinases [20] and GH20 ␤-N-acetylhexosaminidases [21] have indicated that the catalysis of these GH18 and GH20 enzymes proceeds in a substrate-assisted mechanism involving the participation of the 2-acetamide group in the substrate. In this mechanism (Scheme 1), a catalytic residue (Asp or Glu) acts as a general acid to protonate the glycosidic oxygen. Kinetic studies reveal the nature of the enhancement of the transglycosylation efficiency and provide further insights into the transglycosylation mechanism of Endo-M

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