Glycoside Hydrolase Family 54

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• Author: Shinya Fushinobu
• Responsible Curator: Shinya Fushinobu

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Glycoside Hydrolase Family 54
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH54.html

Substrate specificities

This family of glycoside hydrolases contains α-L-arabinofuranosidase (EC 3.2.1.55) and β-xylosidase (EC 3.2.1.37). Most of the family members have eukaryotic (fungal) origin. Several homologous genes are found from Bacterial genomes, but none of their gene products are characterized.

Kinetics and Mechanism

Glycoside hydrolases of family 54 are retaining enzymes. In 1996, many α-L-arabinofuranosidases from fungi, including GH54 arabinofuranosidase B from Aspergillus niger, were shown to use a retaining mechanism by ¹H NMR analysis using p-nitrophenyl α-L-arabinofuranoside and by measurement of glycosyl transfer reactions to methanol [1]. In the 1980's, two α-L-arabinofuranosidases (AF I and AF III) from Monilinia fructigena were shown to be retaining enzymes by various methods including suicide inactivation with ³H-labelled 1-α-L-arabinofuranosylmethyl-3-p-nitrophenyltriazene as well as measurement of glycosyl transfer reactions using p-nitrophenyl α-L-arabinofuranoside and methanol [2, 3], but the genes for these enzymes are not cloned yet. If either AF I or AF III belongs to GH54, one of these studies represents the first stereochemistry determination of this family.

Catalytic Residues

The catalytic residues were firstly estimated by superimposing the active site structure of α-L-arabinofuranosidase B (AkAbfB) from Aspergillus kawachii with a Glycoside Hydrolase Family 51 enzyme, α-L-arabinofuranosidase A from Geobacillus stearothermophilus T-6. Based on the relative positions with an arabinose molecule, Glu221 and Asp297 were estimated as the catalytic nucleophile and the general acid/base, respectively. E221A mutant of this enzyme showed no detectable activity, and E297A mutant showed 10^-3-fold activity of wild type [4].
Kinetic studies using mutant enzymes of α-L-arabinofuranosidase from Trichoderma koningii G-39 confirmed this assignment [5].

Three-dimensional structures

The first solved 3-D structure was α-L-arabinofuranosidase B (AkAbfB) from Aspergillus kawachii IFO 4308 (PDB ID 1wd4 in complex with arabinose) in 2004 [4]. This family has a β-jelly roll fold that is slightly similar to Clan GH-B. The position of the catalytic nucleophile is superimposable with Clan GH-B, whereas the general acid/base is not.

Carbohydrate-Binding Module

Most of the members in GH54 have CBM42 of approximately 160 residues at the C-terminus of GH54 catalytic domains. This module was firstly found as a xylan binding domain [6], and binding to arabinofuranose (present in arabinoxylan) has been subsequently demonstrated [7].

Family Firsts

First sterochemistry determination

Probably ABF B from Aspergillus niger and other fungal enzymes by ¹H NMR spectroscopy and glycosyl transfer reaction measurements [1]. See kinetics and mechanism.

First gene cloning

α-L-Arabinofuranosidase (ABF B) from Aspergillus niger (Uniprot P42255) [8].

First catalytic nucleophile identification

AkAbfB from Aspergillus kawachii by structural comparison and mutation.

First general acid/base residue identification

AkAbfB from Aspergillus kawachii by structural comparison and mutation.

First 3-D structure

AkAbfB from Aspergillus kawachii by X-ray crystallography [4].

References

1. Pitson SM, Voragen AG, and Beldman G. (1996). Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases. FEBS Lett. 1996;398(1):7-11. DOI:10.1016/s0014-5793(96)01153-2 |

2. Fielding, A. H., Sinnott, M. L., Kelly, M. A., and Widdows, D. (1981) Product stereochemistry and some inhibitors of the alpha-arabinofuranosidases of Monilinia fructigena. J. Chem. Soc., Perkin Trans. 1, 1013-1014. doi:10.1039/P19810001013.

3. Kelly MA, Sinnott ML, and Herrchen M. (1987). Purification and mechanistic properties of an extracellular alpha-L-arabinofuranosidase from Monilinia fructigena. Biochem J. 1987;245(3):843-9. DOI:10.1042/bj2450843 |
4. Miyanaga A, Koseki T, Matsuzawa H, Wakagi T, Shoun H, and Fushinobu S. (2004). Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose. J Biol Chem. 2004;279(43):44907-14. DOI:10.1074/jbc.M405390200 |
5. Wan CF, Chen WH, Chen CT, Chang MD, Lo LC, and Li YK. (2007). Mutagenesis and mechanistic study of a glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Trichoderma koningii. Biochem J. 2007;401(2):551-8. DOI:10.1042/BJ20060717 |
6. Nogawa M, Yatsui K, Tomioka A, Okada H, and Morikawa Y. (1999). An alpha-L-arabinofuranosidase from Trichoderma reesei containing a noncatalytic xylan-binding domain. Appl Environ Microbiol. 1999;65(9):3964-8. DOI:10.1128/AEM.65.9.3964-3968.1999 |
7. Miyanaga A, Koseki T, Miwa Y, Mese Y, Nakamura S, Kuno A, Hirabayashi J, Matsuzawa H, Wakagi T, Shoun H, and Fushinobu S. (2006). The family 42 carbohydrate-binding module of family 54 alpha-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose. Biochem J. 2006;399(3):503-11. DOI:10.1042/BJ20060567 |
8. Flipphi MJ, van Heuvel M, van der Veen P, Visser J, and de Graaff LH. (1993). Cloning and characterization of the abfB gene coding for the major alpha-L-arabinofuranosidase (ABF B) of Aspergillus niger. Curr Genet. 1993;24(6):525-32. DOI:10.1007/BF00351717 |