View source for Glycoside Hydrolase Family 116

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* [[Author]]: [[User:Beatrice Cobucci-Ponzano|Beatrice Cobucci-Ponzano]], [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]:  [[User:Marco Moracci|Marco Moracci]]
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<!-- The data in the table below should be updated by the Author/Curator according to current information on the family -->
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{| {{Prettytable}} 
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|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH116'''
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|'''Clan'''    
| GH-O
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
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|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
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| colspan="2" |{{CAZyDBlink}}GH116.html
|}
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== Substrate specificities ==
This family of [[glycoside hydrolases]] was discovered characterising a β-glycosidase from the hyperthermophilic archaeon ''Sulfolobus solfataricus'' <cite>CobucciPonzano2010</cite> and contains mammalian non-lysosomal bile acid β-glucosidase GBA2 (EC [{{EClink}}3.2.1.45 3.2.1.45], also known as glucosylceramidase), β-glucosidase (EC [{{EClink}}3.2.1.21 3.2.1.21]) and β-xylosidase (EC [{{EClink}}3.2.1.37 3.2.1.37]) activities from the three  domains of life. The β-glycosidase from ''S. solfataricus'' (SSO1353) is specific for the gluco- and xylosides β-bound to hydrophobic groups that are hydrolyzed by following a ''[[retaining]]'' reaction mechanism. Human non-lysosomal bile acid β-glucosidase GBA2, is involved in the catabolism of glucosylceramide, which is then converted to sphingomyelin <cite>Boot2007</cite>. A β-N-acetylglucosaminidase from ''S. solfataricus'' (SSO3039) from the same family <cite>Ferrara2013</cite> was shown to act as a bifunctional β-glucosidase/β-''N''-acetylglucosaminidase. Phylogenetic analysis allowed classification of GH116 into three subfamilies <cite>Ferrara2013</cite>, each of which now has an enzyme characterized in detail: subfamily 1 contains GBA2 glucosylceramidase <cite>Boot2007</cite>, subfamily 2 includes SSO3039 <cite>Ferrara2013</cite>, and subfamily 3 contains SSO1353 <cite>CobucciPonzano2010</cite>. The three subfamilies are functionally different and are hypothesized to have evolved from a common ancestor. Common characteristics of family GH116 are the specificity for β-glucosides and the ''[[retaining]]'' reaction mechanism. However, subfamilies 1, 2, and 3, have also specificity for glucosylceramides, ''N''-acetyl-glucosaminides, and xylosides, respectively, and peculiar sensitivity to competitive inhibitors. In fact, GBA2 (subfamily 1) is insensitive to CBE and is inhibited by nM amounts of NB-DNJ <cite>Boot2007</cite>, SSO3039 (subfamily 2) is sensitive to μM and mM concentrations of NB-DNJ and CBE, respectively <cite>Ferrara2013</cite>, whilst SSO1353 (subfamily 3) shows mM sensitivity to both NB-DNJ and CBE <cite>CobucciPonzano2010</cite>.

== Kinetics and Mechanism ==
The enzymes of this family are ''[[retaining]]'' glycoside hydrolases and follow the classical [[Koshland double-displacement mechanism]] <cite>Koshland1953</cite>. The stereochemistry of hydrolysis has been demonstrated by <sup>1</sup>H-<sup>13</sup>C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation reaction of SSO1353 with 4NP-β-Xyl <cite>CobucciPonzano2010</cite>, and by direct observation of the formation of β-glucose in the hydrolysis of PNP β-glucoside by ''Thermoanaerobacterium xylanolyticum'' ''Tx''GH116 β-glucosidase by <sup>1</sup>H NMR spectroscopy <cite>Charoenwattanasatien2016</cite>.

== Catalytic Residues ==
The catalytic residues were identified in the ''S. solfataricus'' β-glycosidase SSO1353 <cite>CobucciPonzano2010</cite>. The [[catalytic nucleophile]] was identified as Glu335 through trapping of the 2-deoxy-2-fluoroglucosyl-enzyme [[intermediate]] and MS/MS analysis. The [[general acid/base]] catalyst role was assigned to Asp462 through mechanistic analysis of a mutant at that position, which included azide rescue experiments.

== Three-dimensional structures ==
The structure of ''T. xylanolyticum'' ''Tx''GH116 β-glucosidase has been reported <cite>Charoenwattanasatien2016</cite>. This structure consists of an N-terminal domain, comprised of a two-sheet β-sandwich, and a C-terminal (α/α)<sub>6</sub> solenoid domain. The catalytic nucleophile and general acid/base are contained within the C-terminal domain. The putative catalytic nucleophile, E441, lies near the end of a loop between the first and second α-helices of the C-terminal domain; the putative catalytic acid/base, D593, lies in a loop between the fifth and sixth helices of the C-terminal domain. A Ca<sup>2+</sup> is bound to a site within the same loop that contains the general acid/base.

== Family Firsts ==
;First stereochemistry determination: ''S. solfataricus'' β-glycosidase SSO1353 by NMR analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation reaction with 4NP-β-Xyl  <cite>CobucciPonzano2010</cite>.
;First catalytic nucleophile identification: ''S. solfataricus'' β-glycosidase  SSO1353 by 2-deoxy-2-fluoroglucose labeling <cite>CobucciPonzano2010</cite>.
;First general acid/base residue identification: ''S. solfataricus'' β-glycosidase SSO1353 by azide rescue with mutant <cite>CobucciPonzano2010</cite>.
;First 3-D structure: ''Thermoanaerobacterium xylanolyticum'' ''Tx''GH116 β-glucosidase <cite>Charoenwattanasatien2016</cite>.

== References ==
<biblio>
#CobucciPonzano2010 pmid=20427274
#Boot2007 pmid=17105727
#Ferrara2013 pmid=24060745
#Koshland1953 Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]
#Charoenwattanasatien2016 pmid=27115290
</biblio>


[[Category:Glycoside Hydrolase Families|GH116]]