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. 2024 Oct;41(4-5):279-289.
doi: 10.1007/s10719-024-10166-7. Epub 2024 Sep 28.

Site-directed mutagenesis leads to the optimized transglycosylation activity of endo-beta-N-acetylglucosaminidase from Trypanosoma brucei

Yi Ding  1   2   3 , Zheng-Hui Chen  1 , Juan Cui  1 , Xin-Yu Ding  1 , Xiao-Dong Gao  4   5   6 , Ning Wang  7   8   9
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Site-directed mutagenesis leads to the optimized transglycosylation activity of endo-beta-N-acetylglucosaminidase from Trypanosoma brucei

Yi Ding et al. Glycoconj J. 2024 Oct.

Abstract

Endo-β-N-acetylglucosaminidases (ENGases) are pivotal enzymes in the degradation and remodeling of glycoproteins, which catalyze the cleavage or formation of β-1,4-glycosidic bond between two N-acetylglucosamine (GlcNAc) residues in N-linked glycan chains. It was investigated that targeted mutations of amino acids in ENGases active site may modulate their hydrolytic and transglycosylation activities. Endo-Tb, the ENGase derived from Trypanosoma brucei, belongs to the glycoside hydrolase family 85 (GH85). Our group previously demonstrated that Endo-Tb exhibits hydrolytic activity toward high-mannose and complex type N-glycans and preliminarily confirmed its transglycosylation potential. In this study, we further optimized the transglycosylation activity of recombinant Endo-Tb by focusing on the N536A, E538A and Y576F mutants. A comparative analysis of their transglycosylation activity with that of the wild-type enzyme revealed that all mutants exhibited enhanced transglycosylation capacity. The N536A mutant exhibited the most pronounced improvement in transglycosylation activity with a significant reduction in hydrolytic activity. It is suggested that Endo-Tb N536A possesses the potential as a tool for synthesizing a wide array of glycoconjugates bearing high-mannose and complex type N-glycans.

Keywords: N-glycan; Trypanosoma brucei; Endo-β-N-acetylglucosaminidases; Site mutation; Transglycosylation.

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