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• Author: Wade Abbott
• Responsible Curator: Alisdair Boraston

Substrate specificities

Endo-beta-N-acetylglucosaminidases (ENGse) are glycoside hydrolases that 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, 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 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 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 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 modified 2-acetamido group of NAG-thiazoline inhibitors [5, 11]. E177 operates as the catalytic acid and donates a protein to the glycosidic oxygen [10].

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: It was suggested that the 2-acetamido group acts as a substrate-borne nucleophile based on transglycosylation observed with a disaccharide oxazoline substrate [12] .

First general acid/base residue identification: The general base residue 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 TIGR4 Endo-D PDB IDs: 2w91 and 2w92 (release date: 2009-01-27). [5].

References

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2. Karamanos Y, Bourgerie S, Barreaud JP, and Julien R. Are there biological functions for bacterial endo-N-acetyl-beta-D-glucosaminidases?. Res Microbiol 1995 Jul-Aug; 146(6) 437-43. pmid:8525060. PubMed HubMed [2]
3. Barreaud JP, Bourgerie S, Julien R, Guespin-Michel JF, and Karamanos Y. An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus. J Bacteriol 1995 Feb; 177(4) 916-20. pmid:7860600. PubMed HubMed [3]
4. Takegawa K, Fujiwara K, Iwahara S, Yamamoto K, and Tochikura T. Effect of deglycosylation of N-linked sugar chains on glucose oxidase from Aspergillus niger. Biochem Cell Biol 1989 Aug; 67(8) 460-4. pmid:2511903. PubMed HubMed [4]
5. Abbott DW, Macauley MS, Vocadlo DJ, and Boraston AB. Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases. J Biol Chem 2009 Apr 24; 284(17) 11676-89. doi:10.1074/jbc.M809663200 pmid:19181667. PubMed HubMed [5]
6. Fujita K, Kobayashi K, Iwamatsu A, Takeuchi M, Kumagai H, and Yamamoto K. Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme. Arch Biochem Biophys 2004 Dec 1; 432(1) 41-9. doi:10.1016/j.abb.2004.09.013 pmid:15519295. PubMed HubMed [6]
7. Li SC, Asakawa M, Hirabayashi Y, and Li Y. Isolation of two endo-beta-N-acetylglucosaminidases from fig latex. Biochim Biophys Acta 1981 Aug 13; 660(2) 278-83. pmid:6793075. PubMed HubMed [7]
8. Ito K, Okada Y, Ishida K, and Minamiura N. Human salivary endo-beta-N-acetylglucosaminidase HS specific for complex type sugar chains of glycoproteins. J Biol Chem 1993 Jul 25; 268(21) 16074-81. pmid:8340428. PubMed HubMed [8]
9. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, and Gerngross TU. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 2006 Sep 8; 313(5792) 1441-3. doi:10.1126/science.1130256 pmid:16960007. PubMed HubMed [9]
10. Umekawa M, Huang W, Li B, Fujita K, Ashida H, Wang LX, and Yamamoto K. Mutants of Mucor hiemalis endo-beta-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J Biol Chem 2008 Feb 22; 283(8) 4469-79. doi:10.1074/jbc.M707137200 pmid:18096701. PubMed HubMed [10]
11. Yin J, Li L, Shaw N, Li Y, Song JK, Zhang W, Xia C, Zhang R, Joachimiak A, Zhang HC, Wang LX, Liu ZJ, and Wang P. Structural basis and catalytic mechanism for the dual functional endo-beta-N-acetylglucosaminidase A. PLoS One 2009; 4(3) e4658. doi:10.1371/journal.pone.0004658 pmid:19252736. PubMed HubMed [11]
12. Fujita M, Shoda S, Haneda K, Inazu T, Takegawa K, and Yamamoto K. A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases. Biochim Biophys Acta 2001 Sep 3; 1528(1) 9-14. pmid:11514092. PubMed HubMed [13]
13. Ling Z, Suits MD, Bingham RJ, Bruce NC, Davies GJ, Fairbanks AJ, Moir JW, and Taylor EJ. 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 May 29; 389(1) 1-9. doi:10.1016/j.jmb.2009.03.050 pmid:19327363. PubMed HubMed [12]