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

Abstract：

: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 the most efficient glycosynthase variant with the highest transglycosylation/hydrolysis ratio, which is similar to the recently reported D184M mutant of Endo-S2, another S. pyogenes endoglycosidase. Kinetic studies of the D233M and D233A mutants of Endo-S, as well as glycosynthase mutants D184M and D184A of Endo-S2, indicated that the enhanced catalytic efficacy of the Asp-to-Met mutants of both enzymes was mainly due to an increased turnover number (increased kcat) for the glycan oxazoline substrate and the significantly enhanced substrate affinity (as judged by the reduced KM value) for the antibody acceptor.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Tong X,Li T,Li C,Wang LX

doi

10.1021/acs.biochem.8b00719

subject

Has Abstract

pub_date

2018-09-04 00:00:00

pages

5239-5246

issue

35

eissn

0006-2960

issn

1520-4995

journal_volume

57

pub_type

杂志文章

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