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Thus far only one member of the family has been characterised, BT1012 from bacteroides thetaiotaomicron. BT1012 displays endo-apiosidase activity targeting α1,2 linked apiose at the base of the sidechains A and B in the complex glycan rhamnogalacturonan ii (RGII) [1]. Cleavage of the RGII backbone must occur before BT1012 can act [1].
Kinetics and Mechanism
GH140 likely uses a, retaining, double displacement mechanism. This is strongly supported by methanolysis experiments where BT1012 was incubated with trisaccharide L-rhamnose-β1,3-D-apiose-α1,2-D-galacturonic acid-O-methyl in the presence of 10 % methanol. This experiment performed with BT1012 generated the product L-rhamnose-β1,3-D-apiose-O-methyl suggesting a retaining mechanism [1]. Glycoside hydrolases that utilise a retaining mechanism, but not those that use an inverting mechanism, can utilise methanol as an external nucleophile and thus generate a methylated product.
Catalytic Residues
The catalytic residues are an aspartate and glutamate located on the top of β-strands 4 and 7, respectively [1]. This could mean GH140 is a distant relative of Clan GH-A enzymes, however in with GH-A the catalytic residue atop of β-strands 4 and 7 are both glutamates. In the absence of a ligand bound complex or more detailed biochemical analysis [2] (preferably both) it is not possible to say which of the catalytic residues is the nucleophile or acid/base.
Three-dimensional structures
GH140 adopts a (β/α)8 , TIM barrel, where a central barrel of eight β strands are encircled by eight α helices [1]. BT1012, the only GH140 structure available, also has a Ig like domain that stacks against the TIM barrel likely providing structural stability, similar to the role of Ig like domains in GH43 enzymes [3, 4].
Family Firsts
First stereochemistry determination
Methanolysis experiments suggest a retaining mechanism [1].
