Glycoside Hydrolase Family 43

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Glycoside Hydrolase Family GH43
Clan	GH-F
Mechanism	inverting
Active site residues	Known
CAZy DB link
https://www.cazy.org/GH43.html

Substrate specificities

The major activities reported for this family of glycoside hydrolases are α-L-arabinofuranosidases [1], endo-α-L-arabinanases (or endo-processive arabinanases) [2, 3] and β-D-xylosidases [4] (for further details see: Absolute configuration: D/L nomenclature). An enzyme with exo α-1,3-galactanase has also been described [5]. A significant number of enzymes in this family display both α-L-arabinofuranosidase and β-D-xylosidase activity using aryl-glycosides as substrates. It is likely that the natural activity of these enzymes is conferred by the leaving-group component of the substrate. Indeed, the arabionofuranosidase activities already reported target very different glycans. Thus, the Bacillus subtilis enzyme arabinoxylan α-L-arabinofuranohydrolase specifically removes arabinofuranose side chains that are linked either α-1,2 or α-1,3 to backbone xylose residues [6], while the arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis will remove an α-1,3-linked arabinofuranose from xylans where the xylose residue is substituted at both α-1,2 and α-1,3 with arabinose [7]. By contrast some arabinofuranosidases are exo-α-1,5-L-arabinanases [8]. It should be noted that in several plant cell wall degrading organisms there has been a dramatic expansion in GH43 family enzymes, which may reflect a more extensive range of specificities than described to date. In light of the sequence and functional diversity of GH43 members, this family has been divided into subfamilies [9].

Kinetics and Mechanism

NMR, deploying arabinan as the substrate, showed that an endo-α-1,5-arabinanase uses an inverting mechanism [10]. However, the first demonstration of an inverting enzyme, which was later shown to be a GH43 β-xylosidase, was by using a linked assay with an anomeric stereospecific D-xylose isomerase [11].

Catalytic Residues

The catalytic general base, an aspartate, the catalytic general acid, a glutamate, and an aspartate that modules the pK_a of the general acid were identified through the crystal structure of Cellvibrio japonicus CjAbn43A, and confirmed by site-directed mutagenesis [12]. Further biochemical proof for the catalytic function of the equivalent residues in a β-xylosidase were obtained by demonstrating a relationship between the activity of the catalytic acid and the pK_a of the leaving group of the substrate. The identity of the catalytic base was achieved by azide rescue of a mutant of this residue [4]. In contrast to many inverting glycoside hydrolases there appears to be a single candidate catalytic general base for the arabinofuranosidases and xylosidases in this family, but this residue is absent in GH43 galactosidase [13].

Three-dimensional structures

The GH43 enzymes display a 'non-velcroed' five-bladed-β-propeller. The propeller is based upon a five-fold repeat of blades composed of four-stranded β-sheets [12]. The substrate-binding surface of Arb43A is in a long surface depression, with the catalytic constellation of carboxylates at its center. The exo-processive activity of the enzyme is conferred by a subtle steric block at the +3 subsite explaining why the enzyme releases, exclusively, arabinotriose [14]. In the arabinofuranosidases and xylosidases the active site comprises a deep pocket and the orientation of the substrate is very different between the enzymes, which contributes to the varied specificities observed across the GH43 landscape [15, 16].

Family Firsts

First sterochemistry determination: Determined for the Bacillus pumilus β-xylosidase using an anomeric specific D-xylose isomerase [17] and determined for an arabinanase by proton NMR [10].
First general base residue identification: Based on mutagensis informed by 3D structural data [12]
First general acid residue identification: Based on mutagensis informed by 3D structural data [12]
First 3-D structure: α-L-arabinanase from Cellvibrio japonicus [12].

References

All Medline abstracts: PubMed