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. (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. 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. (2016). Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2). ACS Chem Biol. 2016;11(7):1891-900. DOI:10.1021/acschembio.6b00192 |