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
http://www.cazy.org/GH120.html

Substrate specificities

Glycoside hydrolases of family GH120 have been reported to possess 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. XylB from Bifidobacterium adolescentis possessed only weak activity on xylobiose, but much greater on higher oligomers from xylotriose through xylohexaose [2]. XylB was shown to act to remove xylose residues from the non-reducing end of xylooligosaccharides and thus is an exo-xylosidase [2]. Both T. saccharolyticum XylC and B. adolescentis XylB can hydrolyze assorted aryl β-xylosides [1, 3]. B. adolescentis XylB showed exceptionally weak activity on p-nitrophenyl-α-L-arabinofuranoside [2]; T. saccharolyticum XylC showed no detectable activity against this substrate [1].

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 transglycosylation [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 [3]. 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 [4]. Mutagenesis of these residues to alanine provided mutant proteins that were catalytically inactive [4]. On the basis of the relative orientation and distance from the anomeric centre, two of these residues were tentatively assigned as the nucleophile (Asp382) and the general acid/base (Glu405) [4]. A more detailed mechanistic study was performed on B. adolescentis XylB [3]; 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 [3]. 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 [3].

Three-dimensional structures

The three-dimensional structure has been solved for T. saccharolyticum XylC [4]. 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. The overall fold displays similarity to members of polysaccharide lyase family 1 (PL1) and glycoside hydrolase family 28 (GH28). Separate complexes of XylC have been reported with Tris (PDB ID 3vst), xylose (PDB ID 3vsv), and xylobiose (PDB ID 3vsu); in all three complexes the ligands bind at a similar location assigned as the active site, which was located at the interface of the two β-strand 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. The XylC-xylose complex revealed a large number of surface binding sites. Aside from the four xylose molecules bound at the putative active site, within the asymmetric unit tetramer, 31 other molecules were bound at sites located at the dimer interfaces within the asymmetric unit, and other surface sites.

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 [4].

First general acid/base residue identification

Assigned for Bifidobacterium adolescentis XylB by chemical rescue kinetics with azide [3].

First 3-D structure

XylC from T. saccharolyticum (e.g. PDB ID 3vsu) [4].

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. Lagaert S, Pollet A, Delcour JA, Lavigne R, Courtin CM, and Volckaert G. (2011). Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides. Appl Microbiol Biotechnol. 2011;92(6):1179-85. DOI:10.1007/s00253-011-3396-y |
3. 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 |
4. 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 |