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Glycoside Hydrolase Family 85

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Glycoside Hydrolase Family GH85
Clan GH-K
Mechanism retaining
Active site residues known
CAZy DB link
http://www.cazy.org/GH85.html


Contents

Substrate specificities

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].
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 [13] .
First general acid/base residue identification
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].

References

  1. Li B, Song H, Hauser S, and Wang LX. A highly efficient chemoenzymatic approach toward glycoprotein synthesis. Org Lett. 2006 Jul 6;8(14):3081-4. DOI:10.1021/ol061056m | PubMed ID:16805557 | HubMed [1]
  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. PubMed ID:8525060 | 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. PubMed ID:7860600 | 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. PubMed ID:2511903 | 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 | PubMed ID:19181667 | 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 | PubMed ID:15519295 | 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. PubMed ID:6793075 | 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. PubMed ID:8340428 | 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 | PubMed ID:16960007 | HubMed [9]
  10. Fujita K, Sato R, Toma K, Kitahara K, Suganuma T, Yamamoto K, and Takegawa K. Identification of the catalytic acid base residue of arthrobacter endo-beta-N-acetylglucosaminidase by chemical rescue of an inactive mutant. J Biochem. 2007 Sep;142(3):301-6. DOI:10.1093/jb/mvm124 | PubMed ID:17567654 | HubMed [14]
  11. 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 | PubMed ID:18096701 | HubMed [10]
  12. 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 | PubMed ID:19252736 | HubMed [11]
  13. 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. PubMed ID:11514092 | HubMed [13]
  14. 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 | PubMed ID:19327363 | HubMed [12]
All Medline abstracts: PubMed | HubMed
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