Mutants of Mucor hiemalis endo-beta-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities

Endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the beta1,4 linkage of N,N'-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175"knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-beta-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.