Glycoside Hydrolase Family 81

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

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Glycoside Hydrolase Family GH81
Clan	none
Mechanism	inverting
Active site residues	not known
CAZy DB link
https://www.cazy.org/GH81.html

Substrate specificities

GH81 family are endo-β(1,3)-glucanases (EC 3.2.1.39) with diverse physiological roles, such as plant biomass degradation, cell cycling, and enzymatic pathogen defense. They are mostly found in bacteria and fungi, particularly abundant in Saccharomyces, and Streptomyces species. Activity has been demonstrated on laminarin [1, 2, 3, 4, 5, 6], curdlan [1, 3, 5, 6], and pachyman [2, 5].

Kinetics and Mechanism

GH81 enzymes follow an inverting mechanism, first shown by ¹H-NMR during the hydrolysis of laminarioligosaccharides [7], and laminarin [2], thus operating by a single-displacement mechanism.

Catalytic Residues

Primary sequence alignments of GH81 reveal a number of highly conserved residues, including two glutamate residues and one aspartate residue which are located in the active site cleft and likely to serve as catalytic residues [3, 4, 8].

Mutagenesis experiments, chemical rescue, and examination of the active site architecture and product conformational itinerary of ligand complexes strongly suggest that one of the two glutamic acids (E542 in GH81 from Bacillus halodurans C-125) acts as the catalytic base by activating a correctly positioned catalytic water, and the aspartic acid (D422 in BhGH81) acts as the catalytic acid [5, 6, 8]. While mutagenesis studies show that mutation of the second glutamic acid (E546 in BhGH81) results in a dramatic reduction in activity [8], there is conflicting evidence for the role of this residue in catalysis. Pluvinage et al. report that this residue is not correctly positioned to assist in catalysis [5], while Zhou et al. proposes that this residue is the catalytic acid, as the distance between the two glutamic acids is ideal for the inverting mechanism displayed by these enzymes [4]. As such, the exact identity of the catalytic residues for GH81 enzymes remains unclear.

Three-dimensional structures

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Family Firsts

First stereochemistry determination

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First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

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References

1. Fontaine T, Hartland RP, Beauvais A, Diaquin M, and Latge JP. (1997). Purification and characterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus. Eur J Biochem. 1997;243(1-2):315-21. DOI:10.1111/j.1432-1033.1997.0315a.x |
2. McGrath CE and Wilson DB. (2006). Characterization of a Thermobifida fusca beta-1,3-glucanase (Lam81A) with a potential role in plant biomass degradation. Biochemistry. 2006;45(47):14094-100. DOI:10.1021/bi061757r |
3. Martín-Cuadrado AB, Fontaine T, Esteban PF, del Dedo JE, de Medina-Redondo M, del Rey F, Latgé JP, and de Aldana CR. (2008). Characterization of the endo-beta-1,3-glucanase activity of S. cerevisiae Eng2 and other members of the GH81 family. Fungal Genet Biol. 2008;45(4):542-53. DOI:10.1016/j.fgb.2007.09.001 |
4. Zhou P, Chen Z, Yan Q, Yang S, Hilgenfeld R, and Jiang Z. (2013). The structure of a glycoside hydrolase family 81 endo-β-1,3-glucanase. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 10):2027-38. DOI:10.1107/S090744491301799X |
5. Pluvinage B, Fillo A, Massel P, and Boraston AB. (2017). Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of β-1,3-Glucan Quaternary Structure. Structure. 2017;25(9):1348-1359.e3. DOI:10.1016/j.str.2017.06.019 |
6. Kumar K, Correia MAS, Pires VMR, Dhillon A, Sharma K, Rajulapati V, Fontes CMGA, Carvalho AL, and Goyal A. (2018). Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase. Int J Biol Macromol. 2018;117:890-901. DOI:10.1016/j.ijbiomac.2018.06.003 |
7. Fliegmann J, Montel E, Djulić A, Cottaz S, Driguez H, and Ebel J. (2005). Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases. FEBS Lett. 2005;579(29):6647-52. DOI:10.1016/j.febslet.2005.10.060 |
8. McGrath CE, Vuong TV, and Wilson DB. (2009). Site-directed mutagenesis to probe catalysis by a Thermobifida fusca beta-1,3-glucanase (Lam81A). Protein Eng Des Sel. 2009;22(6):375-82. DOI:10.1093/protein/gzp015 |