Structural Basis and Catalytic Mechanism for the Dual Functional Endo-β-N-Acetylglucosaminidase A

Jie Yin,Wenpeng Zhang,Zhi-Jie Liu,Hou-Cheng Zhang,Yang Li,Andrzej Joachimiak,Peng Wang,Rongguang Zhang,Lei Li,Lai-Xi Wang,Jing Katherine Song,Neil Shaw,Chengfeng Xia,Bostjan Kobe

doi:10.1371/journal.pone.0004658

Abstract

Endo-β-N-acetylglucosaminidases (ENGases) are dual specificity enzymes with an ability to catalyze hydrolysis and transglycosylation reactions. Recently, these enzymes have become the focus of intense research because of their potential for synthesis of glycopeptides. We have determined the 3D structures of an ENGase from Arthrobacter protophormiae (Endo-A) in 3 forms, one in native form, one in complex with Man3GlcNAc-thiazoline and another in complex with GlcNAc-Asn. The carbohydrate moiety sits above the TIM-barrel in a cleft region surrounded by aromatic residues. The conserved essential catalytic residues – E173, N171 and Y205 are within hydrogen bonding distance of the substrate. W216 and W244 regulate access to the active site during transglycosylation by serving as “gate-keepers”. Interestingly, Y299F mutation resulted in a 3 fold increase in the transglycosylation activity. The structure provides insights into the catalytic mechanism of GH85 family of glycoside hydrolases at molecular level and could assist rational engineering of ENGases.

Highlights

Endo-b-N-acetylgucosaminidases (EC 3.2.1.96) (ENGases) are a class of enzymes that hydrolyze the glycosidic bond - GlcNAc b1,4GlcNAc - present in N-linked sugar chains in glycoproteins and release the N-glycan moiety
The transglycosylation activities in general are relatively low in comparison with their hydrolytic activity, when natural N-glycan or natural glycopeptide is used as the donor substrate
Product hydrolysis is a major issue for this chemoenzymatic approach, as the product formed would turn out to be the substrate of the enzyme

Summary

Introduction

Endo-b-N-acetylgucosaminidases (EC 3.2.1.96) (ENGases) are a class of enzymes that hydrolyze the glycosidic bond - GlcNAc b1,4GlcNAc - present in N-linked sugar chains in glycoproteins and release the N-glycan moiety. These enzymes are key enzymes in the processing event of free oligosaccharides in the cytosol [1]. When the acceptor is a pre-assembled GlcNAc-containing peptide or protein, Endo-A and Endo-M are able to transfer an intact oligosaccharide to the acceptor to form a new glycopeptide or glycoprotein in a single step, making it a highly convergent chemoenzymatic approach [13,14,15]. Product hydrolysis is still a problem when this chemoenzymatic approach is applied to the synthesis of glycoproteins carrying natural N-glycans, as the ENGases can rapidly hydrolyze the natural N-glycans

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Conclusion