Glycoside Hydrolase Family 62

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• Author: Harry Gilbert
• Responsible Curator: Harry Gilbert

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

Substrate specificities

This small family of glycoside hydrolases comprises an equal number of eukaryotic and prokaryotic enzymes. All the characterized enzymes in this family are arabinofuranosidases that specifically cleave either α-1,2 or α-1,3-L-arabinofuranose side chains from xylans [1, 2]. The enzymes will not act on xylose moieties in xylan that are decorated at both O2 and O3 with an arabinose side chain. The GH62 enzyme from Cellvibrio japonius also displays no non-specific arabinofuranosidase activity; for example it does not hydrolyse 4-nitrophenyl α-L-arabinofuranoside. Several of these enzymes contain cellulose-[1] or xylan-[3] binding CBMs.

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 [4] 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

Predicted to be an Asp (general acid) and Glu (general base)

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 [5, 6, 7]. The predicted catalytic general acid, catalytic general base and pKa modulator were also confirmed by mutagenesis data [5, 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.

Family Firsts

First sterochemistry determination

No direct experimental proof but 3D structural information point to an inverting mechanism.

First general acid residue identification

3D structural data in concert with supported by mutagenesis data.

First general base residue identification

3D structural data in concert with supported by mutagenesis data.

First 3-D structure

Several papers in 2014 reveal the 5-fold β-propeller fold.

References

1. 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 |
2. 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 |
3. 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 |
4. 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 |