Glycoside Hydrolase Family 120

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• Author: ^^^Spencer Williams^^^
• Responsible Curator: ^^^Spencer Williams^^^

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Glycoside Hydrolase Family GH120
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH120.html

Substrate specificities

Glycoside hydrolases of family GH120 are β-xylosidases. XylC from Thermoanaerobacterium saccharolyticum hydrolyzed xylobiose and xylotriose [1]. No activity was detected on oat spelt or birch wood xylans. Both T. saccharolyticum XylC and XylB from Bifidobacterium adolescentis can hydrolyze assorted aryl β-xylosides [1, 2].

Kinetics and Mechanism

Incubation of XylC from T. saccharolyticum with 4-nitrophenyl β-xyloside and alcohols including methanol, ethanol and 1-propanol resulted in the formation of the corresponding alkyl glycosides through transglycosidation [1]. The stereochemistry of 4-nitrophenyl β-xyloside hydrolysis catalyzed by XylB from Bifidobacterium adolescentis was monitored by ¹H NMR spectroscopy and revealed the initial formation of the β-anomer of xylose [2]. These data support the assignment of a retaining mechanism to these enzymes and the family, and is consistent with the enzyme utilizing a classical Koshland double-displacement mechanism.

Catalytic Residues

A three-dimensional X-ray structure of T. saccharolyticum XylC highlighted three conserved carboxylate residues, Asp382, Glu353 and Glu405, located near the anomeric carbon of xylose and xylobiose boound in the putative active site [3]. Mutagenesis of these residues to alanine provide mutant proteins that were catalytically inactive [3]. On the basis of the relative orientation and distance from the anomeric centre, two of these residues were tentatively assign as nucleophile (Asp382) and acid/base (Glu405). A more detailed mechanistic study was performed on B. adolescentis XylB. For this protein Glu364, Asp393 and Glu416, correspond to the T. saccharolyticum XylC Glu353, Asp382 and Glu405. Of the alanine mutants at these three positions, the D393A mutant suffered the greatest loss in activity (10⁵-fold) using pNP-Xyl as substrate, which was partially restored in the D393E mutant, consistent with a role as nucleophile. The E416A mutant ..

Three-dimensional structures

The three-dimensional structure has been solved for T. saccharolyticum XylC [3]. The protein consists of a β-strand rich fold, which comprises two domains: a core domain that folds into a right-handed parallel β-helix and a small flanking region that folds into a β-sandwich domain. Separate complexes of XylC have been reported with Tris, xylose and xylobiose; in all three complexes the ligands bind at a similar location assigned as the active site. The active site is formed at the interface of the two domains. Three conserved carboxylic acids, Asp382, Glu353 and Glu405, were identified located near the anomeric carbon of the xylose residue in the xylobiose and D-xylose complexes.

Family Firsts

First stereochemistry determination

Observation of transglycosylation by T. saccharolyticum XylC [1].

First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

XylC from T. saccharolyticum (PDB ID 3vsv) [3].

References

1. Shao W, Xue Y, Wu A, Kataeva I, Pei J, Wu H, and Wiegel J. (2011). Characterization of a novel beta-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485. Appl Environ Microbiol. 2011;77(3):719-26. DOI:10.1128/AEM.01511-10 |
2. Cecchini DA, Fauré R, Laville E, and Potocki-Veronese G. (2015). Biochemical identification of the catalytic residues of a glycoside hydrolase family 120 β-xylosidase, involved in xylooligosaccharide metabolisation by gut bacteria. FEBS Lett. 2015;589(20 Pt B):3098-106. DOI:10.1016/j.febslet.2015.08.012 |
3. Huang CH, Sun Y, Ko TP, Chen CC, Zheng Y, Chan HC, Pang X, Wiegel J, Shao W, and Guo RT. (2012). The substrate/product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485. Biochem J. 2012;448(3):401-7. DOI:10.1042/BJ20121359 |