Glycoside Hydrolase Family 62

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• Author: Harry Gilbert and ^^^Casper Wilken^^^
• Responsible Curator: Harry Gilbert

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Glycoside Hydrolase Family GH62
Clan	GH-F
Mechanism	inverting
Active site residues	Known
CAZy DB link
https://www.cazy.org/GH62.html

Substrate specificities

This small family of glycoside hydrolases comprises both eukaryotic and prokaryotic enzymes. All the characterized enzymes in this family are arabinofuranosidases and the majority act on xylose moieties in xylan and arabinose moieties in arabinan that are single substituted with α-1,2 or α-1,3-L-arabinofuranose side chains [1, 2]. However, a single GH62 enzyme from Pencillium oxalicum exclusively act on the α-1,3-L-arabinofuranose side chains [3, 4]. The GH62 enzymes also display limited non-specific arabinofuranosidase activity; for example the arabinofuranosidases exhibit no [5] or very little [6, 7] activity against 4-nitrophenyl α-L-arabinofuranoside. Several of these enzymes contain carbohydrate binding modules that target cellulose- [5] or xylan- [8].

Kinetics and Mechanism

While the catalytic mechanism of this family have not been formerly determined, likely reflecting the extremely quick rate of mutarotation displayed by arabinose, the enzyme is predicted to display a single displacement or inverting mechanism. This prediction is based on the location of GH62 in clan F, the same clan occupied by GH43, which is an inverting family. Prior to 3D structural data the catalytic residues were predicted from sequence homology with GH43 enzymes, given that both the catalytic mechanism and the catalytic apparatus are conserved in glycoside hydrolase families belonging to the same clan. Thus [9] predicts that the catalytic general acid and general base will be a Glu and Asp, respectively, while a second Asp modulates the pKa of the general acid.

Catalytic Residues

Asp (general acid) and Glu (general base), as suggested by tertiary structures [6, 7, 10] and supported by site-directed mutagenesis and kinetic data [6, 7].

Three-dimensional structures

Based on its location in clan F, enzymes from family GH62s are predicted to display a 5-fold β-propeller fold. This hypothesis was confirmed by three papers published in 2014 [6, 7, 10]. The predicted catalytic general acid, catalytic general base and pKa modulator [9] were also confirmed by mutagenesis data [6, 7]. The active site arabinose-containing pocket opens up into a cleft or channel that binds the xylooligosaccharides and thus the xylan chain. The residues that interact with the polysaccharide backbone were identified [6]. In this respect a conserved tyrosine, present on a mobile loop, was shown to make an important contribution to substrate binding through hydrophobic interactions with the arabinose located in the active site [11].

Family Firsts

First sterochemistry determination

No direct experimental proof but 3D structural information point to an inverting mechanism [6, 7, 10].

First general acid residue identification

3D structural data [6, 7, 10] in concert with supporting mutagenesis data [6, 7].

First general base residue identification

3D structural data [6, 7, 10] in concert with supporting mutagenesis data [6, 7].

First 3-D structure

Several papers in 2014 reveal the 5-fold β-propeller fold [6, 7, 10].

References

5. Kellett LE, Poole DM, Ferreira LM, Durrant AJ, Hazlewood GP, and Gilbert HJ. (1990). Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes. Biochem J. 1990;272(2):369-76. DOI:10.1042/bj2720369 |
6. Maehara T, Fujimoto Z, Ichinose H, Michikawa M, Harazono K, and Kaneko S. (2014). Crystal structure and characterization of the glycoside hydrolase family 62 α-L-arabinofuranosidase from Streptomyces coelicolor. J Biol Chem. 2014;289(11):7962-72. DOI:10.1074/jbc.M113.540542 |
7. Wang W, Mai-Gisondi G, Stogios PJ, Kaur A, Xu X, Cui H, Turunen O, Savchenko A, and Master ER. (2014). Elucidation of the molecular basis for arabinoxylan-debranching activity of a thermostable family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus. Appl Environ Microbiol. 2014;80(17):5317-29. DOI:10.1128/AEM.00685-14 |
8. Dupont C, Roberge M, Shareck F, Morosoli R, and Kluepfel D. (1998). Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains. Biochem J. 1998;330 ( Pt 1)(Pt 1):41-5. DOI:10.1042/bj3300041 |
9. Vincent P, Shareck F, Dupont C, Morosoli R, and Kluepfel D. (1997). New alpha-L-arabinofuranosidase produced by Streptomyces lividans: cloning and DNA sequence of the abfB gene and characterization of the enzyme. Biochem J. 1997;322 ( Pt 3)(Pt 3):845-52. DOI:10.1042/bj3220845 |
10. Siguier B, Haon M, Nahoum V, Marcellin M, Burlet-Schiltz O, Coutinho PM, Henrissat B, Mourey L, O'Donohue MJ, Berrin JG, Tranier S, and Dumon C. (2014). First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies. J Biol Chem. 2014;289(8):5261-73. DOI:10.1074/jbc.M113.528133 |
11. Contesini FJ, Liberato MV, Rubio MV, Calzado F, Zubieta MP, Riaño-Pachón DM, Squina FM, Bracht F, Skaf MS, and Damasio AR. (2017). Structural and functional characterization of a highly secreted α-l-arabinofuranosidase (GH62) from Aspergillus nidulans grown on sugarcane bagasse. Biochim Biophys Acta Proteins Proteom. 2017;1865(12):1758-1769. DOI:10.1016/j.bbapap.2017.09.001 |
12. Pons T, Naumoff DG, Martínez-Fleites C, and Hernández L. (2004). Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68. Proteins. 2004;54(3):424-32. DOI:10.1002/prot.10604 |
13. Wilkens C, Andersen S, Dumon C, Berrin JG, and Svensson B. (2017). GH62 arabinofuranosidases: Structure, function and applications. Biotechnol Adv. 2017;35(6):792-804. DOI:10.1016/j.biotechadv.2017.06.005 |
14. Hu Y, Yan X, Zhang H, Liu J, Luo F, Cui Y, Wang W, and Zhou Y. (2018). Cloning and expression of a novel α-1,3-arabinofuranosidase from Penicillium oxalicum sp. 68. AMB Express. 2018;8(1):51. DOI:10.1186/s13568-018-0577-4 |