Remarkable transglycosylation activity of glycosynthase mutants of endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae.
Endo-β-N-acetylglucosaminidase from Streptococcus pneumoniae (Endo-D) is an endoglycosidase capable of hydrolyzing the Fc N-glycan of intact IgG antibodies after sequential removal of the sialic acid, galactose, and internal GlcNAc residues in the N-glycan. Endo-D also possesses transglycosylation activity with sugar oxazoline as the donor substrate, but the transglycosylation yield is low due to enzymatic hydrolysis of the donor substrate and the product. We report here our study on the hydrolytic and transglycosylation activity of recombinant Endo-D and its selected mutants. We found that Endo-D preferred core-fucosylated N-glycan for hydrolysis but favored nonfucosylated GlcNAc acceptor for transglycosylation. Several mutants showed significantly enhanced transglycosylation efficiency over the wild type enzyme. Two mutants (N322Q and N322A) were identified as typical glycosynthases that demonstrated remarkable transglycosylation activity with only marginal or no product hydrolysis activity. Kinetic studies revealed that the N322Q [corrected]and N322A glycosynthases had much higher catalytic efficiency for glycosylating the nonfucosylated GlcNAc acceptor. In comparison, the N322Q was much more efficient than N322A for transglycosylation. However, N322Q and N322A [corrected] could not take more complex N-glycan oxazoline as substrate for transglycosylation, indicating their strict substrate specificity. The usefulness of the N322Q glycosynthase was exemplified by its application for efficient glycosylation remodeling of IgG-Fc domain.
Institute of Human Virology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Shu-Quan
SQ
Fan
Wei
W
Huang
Lai-Xi
LX
Wang
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005643:Fucose / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000235:genetics*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D007074:Immunoglobulin G; D050505:Mutant Proteins; D005643:Fucose; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008025:Ligases