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Difference between revisions of "Glycoside Hydrolase Family 98"
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|'''Mechanism''' | |'''Mechanism''' | ||
| − | | | + | |Inverting |
|- | |- | ||
|'''Active site residues''' | |'''Active site residues''' | ||
| − | | | + | |Known |
|- | |- | ||
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link''' | |{{Hl2}} colspan="2" align="center" |'''CAZy DB link''' | ||
| Line 29: | Line 29: | ||
== Substrate specificities == | == Substrate specificities == | ||
| − | + | The glycoside hydrolases of this family are endo-β-galactosidases. No other activities have been reported. Family 98 glycoside hydrolases are unique in their specificity towards cleavage of A and B trisaccharides from glycoconjugates <cite> Ashida2005 Higgins2009 Shaikh2009</cite>. The presence of a L-fucose residue on the substrate is essential for activity of some members of this family (EABase, Sp4GH98) <cite> Ashida2005 Higgins2009 Shaikh2009</cite>. The crystal structure of Sp3GH98 suggests that the fucose residue is not required as a specificity determinant in this enzyme <cite>Higgins2009</cite>. | |
| − | + | == Kinetics and Mechanism == | |
| − | + | Family GH98 galactosidases are inverting enzymes, as first shown by NMR monitoring of the Sp4GH98 catalyzed hydrolysis of the LewisY tetrasaccharide <cite>Higgins2009</cite>. EABase was also shown to act through an inverting enzyme by NMR monitoring of the EABase catalysed hydrolysis of an artificial substrate, DNP-A-trisaccharide <cite>Shaikh2009</cite>. These results are contrary to the initial predictions made by Rigden <cite>Rigden2005</cite>. EABase follows normal Michaelis-Menten kinetics <cite>Shaikh2009</cite>. | |
| − | |||
| − | |||
== Catalytic Residues == | == Catalytic Residues == | ||
| − | |||
| + | The catalytic base, an aspartate and glutamate diad, and the catalytic acid, glutamate, were identified through the crystal structures of Sp3GH98 and Sp4GH98 in complex with the A trisaccharide and H disaccharide respectively, and confirmed by site-directed mutagenesis <cite>Higgins2009</cite>. Similar results were obtained through kinetic studies of the corresponding acid and base mutants of EABase with the artificial substrate DNP-A-trisaccharide. Further biochemical proof for the catalytic acid residue was obtained by comparison between the activity of the acid mutant and the pKa of the leaving group of the substrate <cite>Shaikh2009</cite>. | ||
== Three-dimensional structures == | == Three-dimensional structures == | ||
| − | + | ||
| + | The first crystal structures from family 98 were the Sp3GH98 and Sp4GH98 enzymes from ''S. pneumoniae'' in complex with the A trisaccharide and H disaccharide respectively <cite>Higgins2009</cite>. Both structures feature a (α/β)<sub> 8</sub> barrel in the catalytic domain <cite>Higgins2009</cite> | ||
== Family Firsts == | == Family Firsts == | ||
| − | ;First sterochemistry determination: | + | |
| − | ;First catalytic | + | ;First sterochemistry determination: |
| − | ;First | + | |
| − | ;First 3-D structure: | + | ''Streptococcus pneumoniae'' TIGR4 endo-b-galactosidase Sp4GH98 by NMR <cite>Higgins2009</cite>. |
| + | ;First catalytic base identification: | ||
| + | |||
| + | Streptococcus pneumoniae SP3-BS71 endo-b-galactosidase Sp3GH98 <cite>Higgins2009</cite>. | ||
| + | |||
| + | Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 <cite>Higgins2009</cite>. | ||
| + | ;First catalytic acid identification: | ||
| + | Streptococcus pneumoniae SP3-BS71 endo-b-galactosidase Sp3GH98 <cite>Higgins2009</cite>. | ||
| + | |||
| + | Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 <cite>Higgins2009</cite>. | ||
| + | |||
| + | ;First 3-D structure: | ||
| + | Streptococcus pneumoniae SP3-BS71 endo-b-galactosidase Sp3GH98 <cite>Higgins2009</cite>. | ||
| + | |||
| + | Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 <cite>Higgins2009</cite>. | ||
== References == | == References == | ||
<biblio> | <biblio> | ||
| − | # | + | #Ashida2005 pmid=15618227 |
| − | # | + | #Higgins2009 pmid=19608744 |
| − | # | + | #Shaikh2009 pmid=19630404 |
| − | # | + | #Rigden2005 pmid=16212961 |
</biblio> | </biblio> | ||
Revision as of 17:30, 20 October 2009
This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.
- Author: ^^^Fathima Shaikh^^^
- Responsible Curator: ^^^Al Boraston^^^
| Glycoside Hydrolase Family GHnn | |
| Clan | GH-x |
| Mechanism | Inverting |
| Active site residues | Known |
| CAZy DB link | |
| http://www.cazy.org/fam/GHnn.html | |
Substrate specificities
The glycoside hydrolases of this family are endo-β-galactosidases. No other activities have been reported. Family 98 glycoside hydrolases are unique in their specificity towards cleavage of A and B trisaccharides from glycoconjugates [1, 2, 3]. The presence of a L-fucose residue on the substrate is essential for activity of some members of this family (EABase, Sp4GH98) [1, 2, 3]. The crystal structure of Sp3GH98 suggests that the fucose residue is not required as a specificity determinant in this enzyme [2].
Kinetics and Mechanism
Family GH98 galactosidases are inverting enzymes, as first shown by NMR monitoring of the Sp4GH98 catalyzed hydrolysis of the LewisY tetrasaccharide [2]. EABase was also shown to act through an inverting enzyme by NMR monitoring of the EABase catalysed hydrolysis of an artificial substrate, DNP-A-trisaccharide [3]. These results are contrary to the initial predictions made by Rigden [4]. EABase follows normal Michaelis-Menten kinetics [3].
Catalytic Residues
The catalytic base, an aspartate and glutamate diad, and the catalytic acid, glutamate, were identified through the crystal structures of Sp3GH98 and Sp4GH98 in complex with the A trisaccharide and H disaccharide respectively, and confirmed by site-directed mutagenesis [2]. Similar results were obtained through kinetic studies of the corresponding acid and base mutants of EABase with the artificial substrate DNP-A-trisaccharide. Further biochemical proof for the catalytic acid residue was obtained by comparison between the activity of the acid mutant and the pKa of the leaving group of the substrate [3].
Three-dimensional structures
The first crystal structures from family 98 were the Sp3GH98 and Sp4GH98 enzymes from S. pneumoniae in complex with the A trisaccharide and H disaccharide respectively [2]. Both structures feature a (α/β) 8 barrel in the catalytic domain [2]
Family Firsts
- First sterochemistry determination
Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 by NMR [2].
- First catalytic base identification
Streptococcus pneumoniae SP3-BS71 endo-b-galactosidase Sp3GH98 [2].
Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 [2].
- First catalytic acid identification
Streptococcus pneumoniae SP3-BS71 endo-b-galactosidase Sp3GH98 [2].
Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 [2].
- First 3-D structure
Streptococcus pneumoniae SP3-BS71 endo-b-galactosidase Sp3GH98 [2].
Streptococcus pneumoniae TIGR4 endo-b-galactosidase Sp4GH98 [2].
References
- Anderson KM, Ashida H, Maskos K, Dell A, Li SC, and Li YT. (2005). A clostridial endo-beta-galactosidase that cleaves both blood group A and B glycotopes: the first member of a new glycoside hydrolase family, GH98. J Biol Chem. 2005;280(9):7720-8. DOI:10.1074/jbc.M414099200 |
- Higgins MA, Whitworth GE, El Warry N, Randriantsoa M, Samain E, Burke RD, Vocadlo DJ, and Boraston AB. (2009). Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases. J Biol Chem. 2009;284(38):26161-73. DOI:10.1074/jbc.M109.024067 |
- Shaikh FA, Randriantsoa M, and Withers SG. (2009). Mechanistic analysis of the blood group antigen-cleaving endo-beta-galactosidase from Clostridium perfringens. Biochemistry. 2009;48(35):8396-404. DOI:10.1021/bi900991h |
- Rigden DJ (2005). Analysis of glycoside hydrolase family 98: catalytic machinery, mechanism and a novel putative carbohydrate binding module. FEBS Lett. 2005;579(25):5466-72. DOI:10.1016/j.febslet.2005.09.011 |
[[Category:Glycoside Hydrolase Families|GHnnn]]