Discovery of ß-N-acetylglucosaminidases from screening metagenomic libraries and their use as thioglycoligase mutants.

ß-N-Acetylhexosaminidases (HexNAcases) are versatile biocatalysts that cleave terminal N-acetylhexosamine units from various glycoconjugates. Established strategies to generate glycoside-forming versions of the wild type enzymes rely on the mutation of their catalytic residues; however, successful examples of synthetically useful HexNAcase mutants are scarce. In order to expand the range of HexNAcases available as targets for enzyme engineering, we functionally screened a metagenomic library derived from a human gut microbiome. From a pool of hits, we characterized four of the more active candidates by sequence analysis and phylogenetic mapping, and found that they all belonged to CAZy family GH20. After detailed kinetic analysis and characterization of their substrate specificities, active site mutants were generated which resulted in the identification of two new thioglycoligases. BvHex E294A and AsHex E301A catalyzed glycosyl transfer to all three of the 3-, 4- and 6-thio-N-acetylglucosaminides (thio-GlcNAcs) that were tested. Both mutant enzymes also catalyzed glycosyl transfer to a cysteine-containing variant of the model peptide Tab1, with AsHex E301A also transferring GlcNAc onto a thiol-containing protein. This work illustrates how large scale functional screening of expressed gene libraries allows the relatively rapid development of useful new glycoside-forming mutants of HexNAcases, expanding the pool of biocatalysts for carbohydrate synthesis.

Facile Formation of ß-thioGlcNAc Linkages to Thiol-Containing Sugars, Peptides, and Proteins using a Mutant GH20 Hexosaminidase.

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.