Glycoside Hydrolase Family 52

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• Author: ^^^Julie Grondin^^^
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Glycoside Hydrolase Family GH52
Clan	GH-O
Mechanism	retaining
Active site residues	known
CAZy DB link
http://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 catalytic 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 6.5Å separation in the active site consistent with other retaining enzymes

Three-dimensional structures

Figure 1. The dimeric structure of GH52 from Geobacillus thermoglucosidasius in complex with xylobiose (orange)(PDB ID 4C1P). The active site is enclosed by residues from both monomers, restricting this enzyme to exo-hydrolysis via steric hindrance of the catalytic site. Figure from [2].

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 |