Generation and Comparative Kinetic Analysis of New Glycosynthase Mutants from Streptococcus pyogenes Endoglycosidases for Antibody Glycoengineering

Chemoenzymatic glycan remodeling by endoglycosidase-catalyzed deglycosylation and reglycosylation is emerging as an attractive approach for producing homogeneous glycoforms of antibodies, and the success of this approach depends on the discovery of efficient endoglycosidases and their glycosynthase mutants. We report in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes (Endo-S) at the critical Asp-233 (D233) site and evaluation of the hydrolysis and transglycosylation activities of the resulting mutants. We found that in addition to the previously identified D233A and D233Q mutants of Endo-S, most of the Asp-233 mutants discovered here were also glycosynthases that demonstrated glycosylation activity using glycan oxazoline as the donor substrate with diminished hydrolytic activity. The glycosynthase activity of the resultant mutants varied significantly depending on the nature of the amino acid substituents. Among them, the D233M mutant was identified as t...