Glycoside Hydrolase Family 43

• Author: Harry Gilbert
• Responsible Curator: Harry Gilbert

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Glycoside Hydrolase Family GH43
Clan	GH-F
Mechanism	inverting
Active site residues	not known
CAZy DB link
http://www.cazy.org/fam/GH43.html

Substrate specificities

The major activities reported for this family are alpha-L-arabinofuranosidases [1], endo-alpha-L-arabinanases (or endo-processive arabinanases) [2, 3] and beta-D-xylosidases [4]. An enzyme with exo alpha1,3-galactanase has also been described [5]. A significant number of enzymes in this family display both alpha-L-arabinofuranosidase and beta-D-xylosidase activity using aryl-glycosides as substrates. It is likely that the natural activity of these enzymes is conferred by the aglycone component of the substrate. Indeed, the arabionofuranosidase activities already reported target very different glycans. Thus, the Bacillus subtilis enzyme arabinoxylan alpha-L-arabinofuranohydrolase specifically removes arabinofuranose side chains that are linked either alpha1,2 or alpha1,3 to backbone xylose residues [6], while the arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis will remove an alpha1,3-linked arabinofuranose from xylans where the xylose residue is substituted at both alpha1,2 and alpha1,3 with arabinose [7]. By contrast some arabinofuranosidases are exo-alpha1,5-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.

Kinetics and Mechanism

NMR, deploying arabinan as the substrate, showed that an endo-alpha1,5-arabinanase displays a single displacement or inverting mechanism [9].

Catalytic Residues

The catalytic general base, an aspartate, the catalytic general acid, a glutamate, and an aspartate that modules the pKa of the general base were identified through the crystal structure of Cellvibrio japonicus CjAbn43A, and confirmed by site-directed mutagenesis [10]. Further biochemical proof for the catalytic function of the equivalent residues in a beta-xylosidase were obtained by demonstrating a relationship between the activity of the catalytic acid and the pKa of the leaving group of the substrate. The identity of the catalytic base was achieved by azide rescue of a mutant of this ressidue [4]. In contrast to many "inverting" glycoside hydrolases there appears to be a single candidate catalytic general base.

Three-dimensional structures

The GH43 enzymes display a 'non-velcroed' five-bladed-beta-propeller. The propeller is based upon a five-fold repeat of blades composed of four-stranded beta-sheets [10]. 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 [11]. 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 [11, 12].

Family Firsts

First sterochemistry determination

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

alpha-L-Arabinanase from Cellvibrio japonicus [1].

References

1. Nurizzo D, Turkenburg JP, Charnock SJ, Roberts SM, Dodson EJ, McKie VA, Taylor EJ, Gilbert HJ, and Davies GJ. (2002). Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold. Nat Struct Biol. 2002;9(9):665-8. DOI:10.1038/nsb835 |
2. Pitson SM, Voragen AG, and Beldman G. (1996). Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases. FEBS Lett. 1996;398(1):7-11. DOI:10.1016/s0014-5793(96)01153-2 |