Glycoside Hydrolase Family 62

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• Author: Harry Gilbert and ^^^Casper Wilkens^^^
• 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 and α-1,3-L-arabinofuranose side chains [1] with K_cat ranging from 0.3 to 180 s^-1 on wheat arabinoxylan [2, 3, 4]. However, a single GH62 enzyme from Pencillium oxalicum exclusively act on the α-1,3-L-arabinofuranose side chains [5]. The GH62 enzymes also display limited non-specific arabinofuranosidase activity; for example the arabinofuranosidases exhibit no [6] or very little [2, 3] activity against 4-nitrophenyl α-L-arabinofuranoside. Several of these enzymes contain carbohydrate binding modules that target cellulose[6] or xylan[7].

Kinetics and Mechanism

The stereochemical course of arabinose was followed by ¹H NMR during hydrolysis of a 50:50 mixture of XA²XX:XA³XX by Aspergillus nidulans α-L-arabinofuranosidase A, resulting in the release of β-furanose demonstrating that GH62 enzymes are inverting enzymes. Due to fast mutarotation, however, the anomeric signal decreased considerably already after 1 min [4].

Catalytic Residues

Asp (general acid) and Glu (general base), as suggested by tertiary structures [2, 3, 8] and supported by site-directed mutagenesis and kinetic data [2, 3].

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 [2, 3, 8]. The predicted catalytic general acid, catalytic general base and pKa modulator [9] were also confirmed by mutagenesis data [2, 3]. 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 substrate backbone were identified for Streptomyces coelicolor α-L-arabinofuranosidase A (ScAbf62A) in a crystal structure in complex with xylopentaose, which spanned subsite +2R to +4NR [2]. 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 [10]. Remarkably, the xylan main chain bound in two orientations in the crystal structures of ScAbf62A and Streptomyces thermoviolaceus α-L-arabinofuranosidase A, as may be required to position both single α-1,2 and α-1,3-L-arabinofuranose side chains in subsite -1 for productive binding in the active site pocket [2, 3].

Family Firsts

First sterochemistry determination

Determined for Aspergillus nidulans α-L-arabinofuranosidase A by ¹H NMR [4].

First general acid residue identification

3D structural data [2, 3, 8] in concert with supporting mutagenesis data [2, 3].

First general base residue identification

3D structural data [2, 3, 8] in concert with supporting mutagenesis data [2, 3].

First 3-D structure

Several papers in 2014 reveal the 5-fold β-propeller fold [2, 3, 8].

References

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