Glycoside Hydrolase Family 146

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Author: ^^^Jonathon Briggs^^^
Responsible Curator: ^^^Harry Gilbert^^^

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

Substrate specificities

This glycoside hydrolase family has only one assigned activity to date, β-arabinofuranosidase. The founding member of this family, BT0349 from Bacteroides thetaiotaomicron, was shown to cleave both β1,2- and β1,3-linked arabinofuranose side chains present in branched sugar beet arabinan [1].

Kinetics and Mechanism

The only characterised GH146 enzyme, the B. thetaiotaomicron β-arabinofuranosidase BT0349, displays exo-activity on β-linked arabinofuranose by deploying 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 the founding member of GH146, BT0349, were 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

The crystal structure of BT0349, solved using SAD 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). An arabinofuranose is 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 completely conserved with a GH127 β-arabinofuranosidase [2]ref, 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