Glycoside Hydrolase Family 85

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• Author: ^^^Wade Abbott^^^
• Responsible Curator: ^^^Al Boraston^^^

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Glycoside Hydrolase Family GHnn
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
http://www.cazy.org/fam/GHnn.html

    Normal   0               false   false   false      EN-US   X-NONE   X-NONE                                                     MicrosoftInternetExplorer4

Substrate specificities

GH85 enzymes, commonly referred to as Endo-beta-N-acetylglucosaminidases (ENGse) cleave the chitobiose core (GlcNAc-beta-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, and Endo-E), bi- and tri-antennary complex type N-glycans (Endo-F2 and Endo-F3), and both substrates (Endo-M). 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 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

GH85s were originally proposed to utilize a substrate-assisted mechanism resulting in the retention of anomeric configuration on the basis of transglycosylation reactions that deployed oxazoline substrates as donor sugars [10]. Further support was provided by the three-dimensional structure of Endo-A [11] and Endo-D [5] in complex with thiazoline-based inhibitors. NMR spectroscopy was utilized on Endo-D products to demonstrate anomeric retention on cleavage products [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 family 18 and 56 glycoside hydrolases [5].

Catalytic Residues

Exploiting the transglycosylation 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 [10]. 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 2-acetamido group in contact with NAG-thiazoline inhibitors [5, 11]. E177 operates as the catalytic acid and donates a protein to the glycosidic oxygen [10], a role first suggested following the mutagenesis of E174 in Endo-H from Streptomyces plicatus [12].

Family Firsts

First stereochemistry determination: ¹H NMR spectroscopy was used on the products of 3-fluoro-4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside cleavage by Endo-D from S. pneumoniae TIGR4 (SpGH85) [5].

First catalytic nucleophile identification: the 2-acetamido group acting as a nucleophile was suggested by [13] following transglycosylation of a disaccharide oxazoline substrate.

First general acid/base residue identification: catalytic acid was identified by the site-directed mutagenesis of E174 in Endo-H [12]. The “catalytic base” that deprotonates the 2-acetamido group was identified by the site-directed mutagenesis of N175 in Endo-M [10].

First 3-D structure: S. pneumoniae R6 Endo-D PDB ID: 3GBD (release date: 2009-03-17). Unpublished.

References

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14. Ling Z, Suits MD, Bingham RJ, Bruce NC, Davies GJ, Fairbanks AJ, Moir JW, and Taylor EJ. (2009). The X-ray crystal structure of an Arthrobacter protophormiae endo-beta-N-acetylglucosaminidase reveals a (beta/alpha)(8) catalytic domain, two ancillary domains and active site residues key for transglycosylation activity. J Mol Biol. 2009;389(1):1-9. DOI:10.1016/j.jmb.2009.03.050 |

[[Category:Glycoside Hydrolase Families|GH85]]