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• Author: Yuval Shoham
• Responsible Curator: Yuval Shoham

Substrate specificities

The majority of the glycoside hydrolases from this family hydrolyze the glycosidic bond between L-arabinofuranosides side chains of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan. A few enzymes of the family exhibit β-1,4-endoglucanase activity towards carboxymethyl cellulose and xylan [1].

Kinetics and Mechanism

GH51 L-arabinfuranosidases are retaining enzymes and follow a classical Koshland retaining mechanism. Owing to the fast mutarotation and tautomerization rates of arabinose, the stereochemical course of the reaction was monitored in presence of methanol and followed by NMR spectroscopy [2, 3, 4]. Enzymes that have been well studied kinetically include the Geobacillus stearothermophilus T-6 and Thermobacillus xylanilyticus α-L-arabinofuranosidases, for which a detailed kinetic study was performed including kinetics with aryl-α-L-arabinofuranosides bearing various leaving groups, Brønsted plots for the E175A acid-base catalytic residue and azide-rescue for the E294A nucleophilc mutant [3, 4, 5].

Catalytic Residues

The general acid/base was first identified in Thermobacillus xylanilyticus (Glu176) [3] and in Geobacillus stearothermophilus T-6 (Glu175) α-arabinofuranosidases [4] using kinetic analysis, pH dependence profiles, and azide rescue of the catalytic mutant. The catalytic nucleophile was first identified in Geobacillus stearothermophilus α-arabinofuranosidase through detailed kinetic studies for the catalytic mutant including azide rescue.

Three-dimensional structures

Three-dimensional structures for GH51 arabinofuranosidases are available for Geobacillus stearothermophilus [6] Clostridium thermocellum [7] and Thermobacillus xylanilyticus [8]. The enzyme in solution is a hexamer (can be described as a trimer of dimmers) and each monomer is organized into two domains: a ‘clan GH-A’ catalytic (β/α)₈ domain and a 12-stranded β sandwich with a jelly-roll topology.

Family Firsts

First sterochemistry determination

Aspergillus niger and Aspergillus aculeatus α-L-arabinfuranosidases carried out in the presence of 2.5 M methanol and followed by ¹H-NMR spectroscopy [2].

First catalytic nucleophile identification

Geobacillus stearothermophilus α-L-arabinofuranosidase through detailed kinetic studies for the catalytic mutant including azide rescue [5].

First general acid/base residue identification

Thermobacillus xylanilyticus and Geobacillus stearothermophilus T-6 α-L-arabinofuranosidases via detailed kinetic studies for the catalytic mutant including azide rescue [3, 4].

First 3-D structure

Geobacillus stearothermophilus α-L-arabinofuranosidase [6].

References

1. Eckert K and Schneider E. A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases. Eur J Biochem 2003 Sep; 270(17) 3593-602. pmid:12919323. PubMed HubMed [Eckert2003]
2. Pitson SM, Voragen AG, and Beldman G. Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases. FEBS Lett 1996 Nov 25; 398(1) 7-11. pmid:8946944. PubMed HubMed [Pitson1996]
3. Debeche T, Bliard C, Debeire P, and O'Donohue MJ. Probing the catalytically essential residues of the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus. Protein Eng 2002 Jan; 15(1) 21-8. pmid:11842234. PubMed HubMed [Debeche2002]
4. Shallom D, Belakhov V, Solomon D, Gilead-Gropper S, Baasov T, Shoham G, and Shoham Y. The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase. FEBS Lett 2002 Mar 13; 514(2-3) 163-7. pmid:11943144. PubMed HubMed [Shallom2002a]
5. Shallom D, Belakhov V, Solomon D, Shoham G, Baasov T, and Shoham Y. Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase. J Biol Chem 2002 Nov 15; 277(46) 43667-73. doi:10.1074/jbc.M208285200 pmid:12221104. PubMed HubMed [Shallom2002b]
6. Hövel K, Shallom D, Niefind K, Belakhov V, Shoham G, Baasov T, Shoham Y, and Schomburg D. Crystal structure and snapshots along the reaction pathway of a family 51 alpha-L-arabinofuranosidase. EMBO J 2003 Oct 1; 22(19) 4922-32. doi:10.1093/emboj/cdg494 pmid:14517232. PubMed HubMed [Hovel2003]
7. Taylor EJ, Smith NL, Turkenburg JP, D'Souza S, Gilbert HJ, and Davies GJ. Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum. Biochem J 2006 Apr 1; 395(1) 31-7. doi:10.1042/BJ20051780 pmid:16336192. PubMed HubMed [Taylor2006]
8. Paës G, Skov LK, O'Donohue MJ, Rémond C, Kastrup JS, Gajhede M, and Mirza O. The structure of the complex between a branched pentasaccharide and Thermobacillus xylanilyticus GH-51 arabinofuranosidase reveals xylan-binding determinants and induced fit. Biochemistry 2008 Jul 15; 47(28) 7441-51. doi:10.1021/bi800424e pmid:18563919. PubMed HubMed [Paes2008]