Glycoside Hydrolase Family 146

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• Author: Jonathon Briggs
• Responsible Curator: Harry Gilbert

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Glycoside Hydrolase Family GH146
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH146.html

Substrate specificities

Enzymes of this family of glycoside hydrolases exhibit β-arabinofuranosidase activity. The founding member of this family, BT0349 from Bacteroides thetaiotaomicron, cleaves both β1,2- and β1,3-linked arabinofuranose side chains present in branched sugar beet arabinan [1].

Kinetics and Mechanism

BT0349 β-arabinofuranosidase displays exo-activity on β-linked arabinofuranosyl groups and has been proposed to act with a retaining mechanism, based on the positions of the catalytic residues (see below) [1].

Catalytic Residues

The catalytic nucleophile and general acid/base residues of BT0349 are proposed to comprise Cys414 and Glu318, respectively [1]. This is based on the conservation of these residues with the catalytic apparatus of GH127 β-arabinofuranosidases [2].

Three-dimensional structures

Figure 1. The 3D crystal structure of BT0349 showing the N-terminal (α/α)₅ barrel catalytic domain in green and the three β-sandwich domains in cyan, magenta and yellow from the N- to the C-terminus (PDB ID 5OPJ). The arabinose bound in the active site is shown in red and the catalytic resisdues are in blue slate. The zinc in the active is in grey.

The crystal structure of BT0349, solved using single-wavelength anomalous dispersion methods to a resolution of 2.1 A, revealed a four-domain structure. The N-terminal catalytic domain comprises an (α/α)₅ barrel followed by three β-sandwich domains (1, 2 & 3). Arabinofuranose was present in the active site pocket of the catalytic domain, while a zinc atom is coordinated by three cysteine residues and a glutamate in the same domain [1]. The catalytic apparatus is conserved with a GH127 β-arabinofuranosidase [2]; however, the GH127 enzyme lacks β-sandwich domain 3, which is positioned over the active site, effectively burying the bound arabinofuranose. GH127 enzymes were shown to cleave glycosidic bonds with retention of anomeric configuration [3] and, given the conservation of the catalytic apparatus in GH127 and GH146 members, enzymes in both families were proposed to hydrolyse arabinofururanosidic bonds through a retaining mechanism [1].

Family Firsts

First stereochemistry determination

Retaining mechanism of the B. thetaiotaomicron β-arabinofuranosidase, BT0349 [1], based on conservation of the catalytic apparatus with retaining GH127 enzymes [2, 3].

First catalytic nucleophile identification

Cys414 of the B. thetaiotaomicron β-arabinofuranosidase, BT0349, based on mutagenesis data, 3D structure [1] and conservation with the catalytic nucleophile of GH127 enzymes [2].

First general acid/base residue identification

Glu318 of the B. thetaiotaomicron β-arabinofuranosidase, BT0349, based on mutagenesis data, 3D structure [1]and conservation with the catalytic nucleophile of GH127 enzymes [2].

First 3-D structure

The B. thetaiotaomicron β-arabinofuranosidase, BT0349. The enzyme contains an N-terminal catalytic domain that folds into a (α/α)₅ barrel, which is followed by three β-sandwich domains.

References

1. Luis AS, Briggs J, Zhang X, Farnell B, Ndeh D, Labourel A, Baslé A, Cartmell A, Terrapon N, Stott K, Lowe EC, McLean R, Shearer K, Schückel J, Venditto I, Ralet MC, Henrissat B, Martens EC, Mosimann SC, Abbott DW, and Gilbert HJ. (2018) Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides. Nat Microbiol. 3, 210-219. DOI:10.1038/s41564-017-0079-1 |
2. Ito T, Saikawa K, Kim S, Fujita K, Ishiwata A, Kaeothip S, Arakawa T, Wakagi T, Beckham GT, Ito Y, and Fushinobu S. (2014) Crystal structure of glycoside hydrolase family 127 β-l-arabinofuranosidase from Bifidobacterium longum. Biochem Biophys Res Commun. 447, 32-7. DOI:10.1016/j.bbrc.2014.03.096 |
3. Fujita K, Takashi Y, Obuchi E, Kitahara K, and Suganuma T. (2014) Characterization of a novel β-L-arabinofuranosidase in Bifidobacterium longum: functional elucidation of a DUF1680 protein family member. J Biol Chem. 289, 5240-9. DOI:10.1074/jbc.M113.528711 |