Glycoside Hydrolase Family 116

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• Author: Beatrice Cobucci-Ponzano, Spencer Williams
• Responsible Curator: Marco Moracci

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Glycoside Hydrolase Family GH116
Clan	GH-O
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 mammalian non-lysosomal bile acid β-glucosidase GBA2 (EC 3.2.1.45, also known as glucosylceramidase), 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. Human non-lysosomal bile acid β-glucosidase GBA2, is involved in the catabolism of glucosylceramide, which is then converted to sphingomyelin [2]. A β-N-acetylglucosaminidase from S. solfataricus (SSO3039) from the same family [3] was shown to act as a bifunctional β-glucosidase/β-N-acetylglucosaminidase. Phylogenetic analysis allowed classification of GH116 into three subfamilies [3], each of which now has an enzyme characterized in detail: subfamily 1 contains GBA2 glucosylceramidase [2], subfamily 2 includes SSO3039 [3], and subfamily 3 contains SSO1353 [1]. 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 [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], and by direct observation of the formation of β-glucose in the hydrolysis of PNP β-glucoside by Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase by ¹H NMR spectroscopy [5].

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

The structure of T. xylanolyticum TxGH116 β-glucosidase has been reported [5]. This structure consists of an N-terminal domain, comprised of a two-sheet β-sandwich, and a C-terminal (α/α)₆ 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 Ca²⁺ 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 [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

Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase [5].

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 |
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 |
3. Ferrara MC, Cobucci-Ponzano B, Carpentieri A, Henrissat B, Rossi M, Amoresano A, and Moracci M. 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 Jan;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. DOI:10.1111/j.1469-185X.1953.tb01386.x
5. Charoenwattanasatien R, Pengthaisong S, Breen I, Mutoh R, Sansenya S, Hua Y, Tankrathok A, Wu L, Songsiriritthigul C, Tanaka H, Williams SJ, Davies GJ, Kurisu G, and Cairns JR. Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2). ACS Chem Biol. 2016 Jul 15;11(7):1891-900. DOI:10.1021/acschembio.6b00192 |