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Endo-β-N-acetylglucosaminidases (ENGse) are glycoside hydrolases that cleave the chitobiose core (GlcNAc-β-1,4-GlcNac) of N-linked glycans. Examples of ENGases have been shown to be active on high-mannose type N-glycans (Endo-H, Endo-A, Endo-Fsp, Endo-F1, Endo-D and Endo-E), bi- and tri-antennary complex type N-glycans (Endo-F2 and Endo-F3), and both substrates (Endo-M) and belong to glycoside hydrolase families 18 and 85. Although specificity appears to be primarily determined by the oligosaccharide glycone [1], there is evidence that structural features within the carbohydrate-protein aglycone region (GlcNAc-Asn) may also play a role in substrate recognition. GH85s, represented by Endo-D, Endo-A, and Endo-M, are broadly distributed in nature having been described in bacteria [2, 3, 4, 5], fungi [6], plants [7] and animals [8]. In several cases, including Endo-A from Arthrobacter protophormiae (ApGH85) and Endo-M from Mucor hiemalis (MhGH85), ENGases have been shown to catalyze transglycosylation reactions, making them useful candidates in the bioengineering of glycoproteins [1] and biologic pharmaceuticals [9].
Kinetics and Mechanism
Enzymes of family GH85 are retaining enzymes and are proposed to utilize neighboring group participation in a mechanism involving substrate-assisted catalysis by the 2-acetamido group of the sugar. This mechanism was proposed on the basis of the identification of a highly conserved catatlytic acid-base E173 in Endo-A [10] and transglycosylation reactions that deployed oxazoline substrates as donor sugars [11]. Further support was provided by the three-dimensional structure of Endo-A [12] and Endo-D [5] in complex with thiazoline-based inhibitors. NMR spectroscopy was used to monitor the Endo-D catalyzed cleavage of a synthetic aryl glycoside to demonstrate retention of the anomeric configuration [5]. GH85s appear to deploy a rare form of substrate-assisted catalysis as a candidate asparagine, operating in an imidic tautomer form, facilitates a “proton shuttle” that results in acid-base catalysis of the glycosidic bond, a role similar to the catalytic aspartates in Glycoside Hydrolase Family 18 and Glycoside Hydrolase Family 56 [5].
Catalytic Residues
Exploiting the transglycosylase capabilities of Endo-M from M. hiemalis, three residues were identified by site directed mutagenesis to be central to the catalytic reaction: N175, E177, and Y217 [11]. Mutation of the tyrosine to phenylalanine diminished hydrolytic capability but enhanced transglycosylation. The role of N175 was demonstrated to be fundamental for hydrolysis as substitution with alanine ablated hydrolysis; however, transglycosylation could be performed using oxazoline substrates. Interactions between homologous asparagines residues in Endo-A (N171) and Endo-D (N335) were confirmed by structural studies, which observed each in contact with the modified 2-acetamido group of NAG-thiazoline inhibitors [5, 12]. E177 operates as the catalytic acid and donates a protein to the glycosidic oxygen [11], which is a conserved role with E173 of Endo-A [10].
Family Firsts
First stereochemistry determination
1H NMR spectroscopy was used on the products of 3-fluoro-4-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside cleavage by Endo-D from S. pneumoniae TIGR4 (SpGH85) [5].
It was suggested that the 2-acetamido group acts as a substrate-borne nucleophile based on transglycosylation observed with a disaccharide oxazoline substrate [13] .
The residue that protonates the glycosidic oxygen and activates the incoming water was identified by the site-directed mutagenesis and azide/sodium formate rescue of E173 in Endo-A [10]; and confirmed by the structure of Endo-D (E337) in complex with NAG-thiazoline [5].
First 3-D structure
S. pneumoniae TIGR4 Endo-D PDB IDs: 2w91 and 2w92 (release date: 2009-01-27)[5].
