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 have been reported to possess on β-xylosidase activity. XylC from Thermoanaerobacterium saccharolyticum hydrolyzed xylobiose and xylotriose, to afford xylose [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 bound 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 assigned as nucleophile (Asp382) and acid/base (Glu405) [3]. A more detailed mechanistic study was performed on B. adolescentis XylB [2]; for this protein Glu364, Asp393 and Glu416 correspond to Glu353, Asp382 and Glu405 in T. saccharolyticum XylC. Kinetic analysis of the alanine mutants at these three positions showed that 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 for Asp393 as nucleophile, and matching the role assigned for Asp382 in T. saccharolyticum XylC [2]. Glu416 was assigned as acid/base on the basis of a range of kinetic experiments including effects upon rate for mutants at this position for aryl xylosides with different leaving group abilities, and through chemical rescue of catalytic activity in the presence of azide [2].

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

Assigned for T. saccharolyticum XylC on the basis of structural analysis, and mutagenesis [3].

First general acid/base residue identification

Assigned for Bifidobacterium adolescentis XylB by chemical rescue kinetics with azide [2].

First 3-D structure

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

References

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