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

Substrate specificities

This family of glycoside hydrolases was recently discovered characterising a new 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]. SSO1353 has substrate specificity and inhibitor sensitivity slightly different from those of GBA2. In fact, the archaeal enzyme can hydrolyze both aryl β-gluco and β-xylosides and it is inhibited by both N-butyldeoxynojirimycin (NB-DNJ) and conduritol β-epoxide (CBE) [1]. Instead, GBA2 is inactive on methylumbellyferyl-β-D-xylopyranoside and is relatively insensitive to CBE [2].

Kinetics and Mechanism

The enzymes of this family are retaining glycoside hydrolases and follow the classical Koshland double-displacement mechanism [3]. 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 [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 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 by 2-deoxy-2-fluoroglucose labeling [1].

First general acid/base residue identification

S. solfataricus β-glycosidase 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. 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 Jul 2; 285(27) 20691-703. doi:10.1074/jbc.M109.086470 pmid:20427274. PubMed HubMed [PMID20427274]
2. Boot RG, Verhoek M, Donker-Koopman W, Strijland A, van Marle J, Overkleeft HS, Wennekes T, and Aerts JM. Identification of the non-lysosomal glucosylceramidase as beta-glucosidase 2. J Biol Chem 2007 Jan 12; 282(2) 1305-12. doi:10.1074/jbc.M610544200 pmid:17105727. PubMed HubMed [PMID17105727]
3. Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436.

   [Koshland]