Glycoside Hydrolase Family 116

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• Author: ^^^Beatrice Cobucci-Ponzano^^^
• Responsible Curator: ^^^Marco Moracci^^^

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Glycoside Hydrolase Family GH116
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH116.html

Substrate specificities

This family of glycoside hydrolases was discovered characterising a β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus [1] and contains acid β-glucosidase (EC 3.2.1.45), β-glucosidase (EC 3.2.1.21) and β-xylosidase (EC 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. SSO1353 is distantly related to the human non-lysosomal bile acid β-glucosidase GBA2, also known as glucocerebrosidase, involved in the catabolism of glucosylceramide, which is then converted to sphingomyelin [2]. Recently, a new β-N-acetylglucosaminidase from S. solfataricus (SSO3039) from the same family [3] was characterized, demonstrating that it is a bifunctional β-glucosidase/β-N-acetylglucosaminidase. The phylogenetic analysis shows that GH116 can be separated in three subfamilies [3], each of which now has an enzyme characterized in detail: subfamily 1 contains GBA2 [2], subfamily 2 includes SSO3039 [3], and subfamily 3 contains SSO1353 [1]. The three subfamilies are functionally different and have evolved from a common ancestor. Common characteristics of family GH116 are the specificity for β-O-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 [2], SSO3039 (subfamily 2) is sensitive to μM and mM concentrations of NB-DNJ and CBE, respectively [3], whilst SSO1353 (subfamily 3) shows mM sensitivity to both NB-DNJ and CBE [1].

Kinetics and Mechanism

 The enzymes of this family are retaining glycoside hydrolases and follow the classical Koshland double-displacement mechanism [4]. The stereochemistry of hydrolysis has been demonstrated by 1H-13C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation reaction of SSO1353 with 4NP-β-Xyl [1].

Catalytic Residues

 The catalytic residues were identified in the S. solfataricus β-glycosidase SSO1353 [1]. 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

 There is currently no 3-D structure representative for GH116 (see GH116 at CAZy DB).

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 [1].

First catalytic nucleophile identification

S. solfataricus β-glycosidase SSO1353 by 2-deoxy-2-fluoroglucose labeling [1].

First general acid/base residue identification

S. solfataricus β-glycosidase SSO1353 by azide rescue with mutant [1].

First 3-D structure

Presently unknown (see GH116 at CAZy DB).

References

1. Cobucci-Ponzano B, Aurilia V, Riccio G, Henrissat B, Coutinho PM, Strazzulli A, Padula A, Corsaro MM, Pieretti G, Pocsfalvi G, Fiume I, Cannio R, Rossi M, and Moracci M. (2010). A new archaeal beta-glycosidase from Sulfolobus solfataricus: seeding a novel retaining beta-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase GBA2. J Biol Chem. 2010;285(27):20691-703. DOI:10.1074/jbc.M109.086470 |
2. Boot RG, Verhoek M, Donker-Koopman W, Strijland A, van Marle J, Overkleeft HS, Wennekes T, and Aerts JM. (2007). Identification of the non-lysosomal glucosylceramidase as beta-glucosidase 2. J Biol Chem. 2007;282(2):1305-12. DOI:10.1074/jbc.M610544200 |
3. Ferrara MC, Cobucci-Ponzano B, Carpentieri A, Henrissat B, Rossi M, Amoresano A, and Moracci M. (2014). The identification and molecular characterization of the first archaeal bifunctional exo-β-glucosidase/N-acetyl-β-glucosaminidase demonstrate that family GH116 is made of three functionally distinct subfamilies. Biochim Biophys Acta. 2014;1840(1):367-77. DOI:10.1016/j.bbagen.2013.09.022 |

4. Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436.