Glycoside Hydrolase Family 89

• Author: ^^^Elizabeth Ficko-Blean^^^
• Responsible Curator: ^^^Alisdair Boraston^^^

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Glycoside Hydrolase Family GH89
Clan	none
Mechanism	Retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH89.html

Substrate specificities

The family 89 glycoside hydrolases are active as α-N-acetylglucosaminidases [1] [2]. The human lysosomal enzyme, NAGLU, is involved in the degradation of heparan sulfate. Mutations in this enzyme can cause a devastating disease called Sanfilippo syndrome type B which is also called mucopolysaccharidosis IIIB.

Kinetics and Mechanism

Mechanistic and structural data is available on CpGH89, a family 89 glycoside hydrolase produced by Clostridium perfringens. CpGH89 uses a double displacement mechanism to hydrolyze the glycosidic bond which results in retention of stereochemistry at the anomeric carbon.

Catalytic Residues

Two catalytically important glutamate residues have been identified in CpGH89, Glu483 and Glu601. These residues are between 6.1-6.7Å apart which is consistent with a retaining catalytic mechanism. Mutation of Glu601 to an alanine results in an apparent abolishment of activity suggesting this residue is active as the catalytic nucleophile. Glu601 resides below the A-face of the sugar ring and is 2.8-3.1Å from C1 and appears suitably placed for nucleophilic attack on the anomeric carbon. Mutation of Glu483 to alanine results in much less severe impairments in catalysis suggesting this residue is active as the catalytic acid/base residue. Glu483 is ~3.6Å from C1 and appears to be positioned in such a way that it would be capable of forming a hydrogen bond with the glycosidic oxygen of the substrate.

Three-dimensional structures

The three dimensional structure is available for CpGH89, see pdbs 2VC9, 2VCA, 2VCB and 2VCC [1]. CpGH89 is a multi-modular protein and quite large (2095 amino acids). Only residues 26-916 were crystallized. The N-terminal domain (residues 26-155) forms a β-sandwich fold and is shares sequence identity to the family 32 carbohydrate-binding modules (CBMs). This module is tightly packed against the rest of the protein through a number of hydrophobic and hydrogen bonding interactions. The catalytic region is comprised of a small mixed α/β domain (residues 170-280), a decorated (α/β)₈ core (residues 280-620), and an all α-helical domain (residues 621-916).

Family Firsts

First sterochemistry determination

¹H NMR spectroscopy reveals that CpGH89 acts with retention of stereochemistry [1].

First catalytic nucleophile identification

Catalytic nucleophile was revealed by site directed mutagenesis on CpGH89 Glu601 [1].

First general acid/base residue identification

The general acid/base was revealed by site directed mutagenesis on CpGH89 Glu483 [1].

First 3-D structure

see pdbs 2VC9, 2VCA, 2VCB and 2VCC [1].

References

1. Ficko-Blean E, Stubbs KA, Nemirovsky O, Vocadlo DJ, and Boraston AB. (2008). Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A. 2008;105(18):6560-5. DOI:10.1073/pnas.0711491105 |
2. Weber B, Hopwood JJ, and Yogalingam G. (2001). Expression and characterization of human recombinant and alpha-N-acetylglucosaminidase. Protein Expr Purif. 2001;21(2):251-9. DOI:10.1006/prep.2000.1361 |
3. Yogalingam G and Hopwood JJ. (2001). Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic, clinical, and biological implications. Hum Mutat. 2001;18(4):264-81. DOI:10.1002/humu.1189 |
4. Weber B, Guo XH, Kleijer WJ, van de Kamp JJ, Poorthuis BJ, and Hopwood JJ. (1999). Sanfilippo type B syndrome (mucopolysaccharidosis III B): allelic heterogeneity corresponds to the wide spectrum of clinical phenotypes. Eur J Hum Genet. 1999;7(1):34-44. DOI:10.1038/sj.ejhg.5200242 |
5. Ryazantsev S, Yu WH, Zhao HZ, Neufeld EF, and Ohmi K. (2007). Lysosomal accumulation of SCMAS (subunit c of mitochondrial ATP synthase) in neurons of the mouse model of mucopolysaccharidosis III B. Mol Genet Metab. 2007;90(4):393-401. DOI:10.1016/j.ymgme.2006.11.006 |
6. Zheng Y, Ryazantsev S, Ohmi K, Zhao HZ, Rozengurt N, Kohn DB, and Neufeld EF. (2004). Retrovirally transduced bone marrow has a therapeutic effect on brain in the mouse model of mucopolysaccharidosis IIIB. Mol Genet Metab. 2004;82(4):286-95. DOI:10.1016/j.ymgme.2004.06.004 |
7. Li HH, Zhao HZ, Neufeld EF, Cai Y, and Gómez-Pinilla F. (2002). Attenuated plasticity in neurons and astrocytes in the mouse model of Sanfilippo syndrome type B. J Neurosci Res. 2002;69(1):30-8. DOI:10.1002/jnr.10278 |
8. Yu WH, Zhao KW, Ryazantsev S, Rozengurt N, and Neufeld EF. (2000). Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B. Mol Genet Metab. 2000;71(4):573-80. DOI:10.1006/mgme.2000.3095 |
9. Zhao KW and Neufeld EF. (2000). Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells. Protein Expr Purif. 2000;19(1):202-11. DOI:10.1006/prep.2000.1230 |
10. Li HH, Yu WH, Rozengurt N, Zhao HZ, Lyons KM, Anagnostaras S, Fanselow MS, Suzuki K, Vanier MT, and Neufeld EF. (1999). Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding alpha-N-acetylglucosaminidase. Proc Natl Acad Sci U S A. 1999;96(25):14505-10. DOI:10.1073/pnas.96.25.14505 |
11. Schmidtchen A, Greenberg D, Zhao HG, Li HH, Huang Y, Tieu P, Zhao HZ, Cheng S, Zhao Z, Whitley CB, Di Natale P, and Neufeld EF. (1998). NAGLU mutations underlying Sanfilippo syndrome type B. Am J Hum Genet. 1998;62(1):64-9. DOI:10.1086/301685 |
12. Zhao HG, Li HH, Bach G, Schmidtchen A, and Neufeld EF. (1996). The molecular basis of Sanfilippo syndrome type B. Proc Natl Acad Sci U S A. 1996;93(12):6101-5. DOI:10.1073/pnas.93.12.6101 |
13. Ohmi K, Kudo LC, Ryazantsev S, Zhao HZ, Karsten SL, and Neufeld EF. (2009). Sanfilippo syndrome type B, a lysosomal storage disease, is also a tauopathy. Proc Natl Acad Sci U S A. 2009;106(20):8332-7. DOI:10.1073/pnas.0903223106 |
14. Beesley CE, Jackson M, Young EP, Vellodi A, and Winchester BG. (2005). Molecular defects in Sanfilippo syndrome type B (mucopolysaccharidosis IIIB). J Inherit Metab Dis. 2005;28(5):759-67. DOI:10.1007/s10545-005-0093-y |
15. Yogalingam G, Weber B, Meehan J, Rogers J, and Hopwood JJ. (2000). Mucopolysaccharidosis type IIIB: characterisation and expression of wild-type and mutant recombinant alpha-N-acetylglucosaminidase and relationship with sanfilippo phenotype in an attenuated patient. Biochim Biophys Acta. 2000;1502(3):415-25. DOI:10.1016/s0925-4439(00)00066-1 |