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

• Published in: Glycoconjugate journal Vol. 41; no. 4-5; pp. 279 - 289
• Main Authors: Ding, Yi, Chen, Zheng-Hui, Cui, Juan, Ding, Xin-Yu, Gao, Xiao-Dong, Wang, Ning
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.10.2024; Springer Nature B.V
• Subjects: Amino acids; Biochemistry; Biomarkers; Biomedical and Life Sciences; Comparative analysis; Enzymes; Gene amplification; Glycoconjugates; Glycoproteins; Glycoside hydrolase; Glycosides; Glycosylation; Laboratories; Life Sciences; Mannose; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - chemistry; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - genetics; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - metabolism; Mutagenesis; Mutagenesis, Site-Directed - methods; Mutants; Mutation; N-Acetylglucosamine; N-Acetylglucosaminidase; N-glycans; Pathology; Plasmids; Polysaccharides; Proteins; Protozoa; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Site-directed mutagenesis; Streptococcus infections; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology; Trypanosoma brucei brucei - genetics; Tuberculosis; Transglycosylation; acetylglucosaminidases; glycan; Endo-β; Site mutation; N-glycan; Endo-β-N-acetylglucosaminidases; Trypanosoma brucei
• ISSN: 0282-0080; 1573-4986; 1573-4986
• DOI: 10.1007/s10719-024-10166-7

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. Graphical Abstract