Glycoside Hydrolase Family 52

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• Author: ^^^Julie Grondin^^^
• Responsible Curator:

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Glycoside Hydrolase Family GH52
Clan	GH-O
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH52.html

Substrate specificities

GH52 enzymes are bacterial exo-β-xylosidases (EC 3.2.1.37), which cleave xylose from the nonreducing end of xylooligosaccharides. Activity has been demonstrated on pNP-β-d-xylopyranoside [1, 2], xylobiose [2], xylotriose [2].

Kinetics and Mechanism

GH52 are retaining enzymes, proceeding via a classical Koshland double-displacement mechanism [3]. This was first shown by ¹H-NMR in the cleavage of pNP-β-D-xylopyranoside by XynB2 from Bacillus stearothermophilus T-6 [1].

Catalytic Residues

Site-directed mutagenesis, chemical rescue, and kinetic profiling of XynB2 from Bacillus stearothermophilus T-6 identified E335 as the nucleophile, and D495 as the general acid/base [1, 4]. These results were further confirmed following the structural analysis of GH52 from Geobacillus thermoglucosidasius [2], their 6.5A separation in the active site consistent with other retaining enzymes

Three-dimensional structures

The structure of GH52 consists of an N-terminal β-sandwich domain and a C-terminal (a/a)₆ barrel domain, classifying these enzymes into the GH-O clan.

The exo-acting mode of action of GH52 is reflected in the topology of the active site. The enzyme acts as a dimer in solution [1, 2], with interactions between monomers forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy ⁴H₃ half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site [2].

Family Firsts

First stereochemistry determination

XynB2 from Bacillus stearothermophilus T-6 by ¹H-NMR for the hydrolysis of pNP-β-D-xylopyranoside [1].

First catalytic nucleophile identification

XynB2 from Bacillus stearothermophilus T-6 by site-directed mutagenesis and chemical rescue [4].

First general acid/base residue identification

XynB2 from Bacillus stearothermophilus T-6 by site-directed mutagenesis, chemical rescue, and pH profiling [4].

First 3-D structure

GH52 from Geobacillus thermoglucosidasius NBRC 107763 [2].

References

1. Bravman T, Zolotnitsky G, Shulami S, Belakhov V, Solomon D, Baasov T, Shoham G, and Shoham Y. (2001). Stereochemistry of family 52 glycosyl hydrolases: a beta-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme. FEBS Lett. 2001;495(1-2):39-43. DOI:10.1016/s0014-5793(01)02360-2 |
2. Espina G, Eley K, Pompidor G, Schneider TR, Crennell SJ, and Danson MJ. (2014). A novel β-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds. Acta Crystallogr D Biol Crystallogr. 2014;70(Pt 5):1366-74. DOI:10.1107/S1399004714002788 |

3. 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

4. Bravman T, Belakhov V, Solomon D, Shoham G, Henrissat B, Baasov T, and Shoham Y. (2003). Identification of the catalytic residues in family 52 glycoside hydrolase, a beta-xylosidase from Geobacillus stearothermophilus T-6. J Biol Chem. 2003;278(29):26742-9. DOI:10.1074/jbc.M304144200 |