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Glycoside Hydrolase Family 43

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Glycoside Hydrolase Family GH43
Clan GH-F
Mechanism inverting
Active site residues Known
CAZy DB link

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.

Kinetics and Mechanism

NMR, deploying arabinan as the substrate, showed that an endo-α-1,5-arabinanase uses an inverting mechanism [9]. 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 [10].

Catalytic Residues

The catalytic general base, an aspartate, the catalytic general acid, a glutamate, and an aspartate that modules the pKa of the general acid were identified through the crystal structure of Cellvibrio japonicus CjAbn43A, and confirmed by site-directed mutagenesis [11]. 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 pKa 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 [12].

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 [11]. 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 [13]. 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 [14, 15].

Family Firsts

First sterochemistry determination
Determined for the Bacillus pumilus β-xylosidase using an anomeric specific D-xylose isomerase [16] and determined for an arabinanase by proton NMR [9].
First general base residue identification
Based on mutagensis informed by 3D structural data [11]
First general acid residue identification
Based on mutagensis informed by 3D structural data [11]
First 3-D structure
α-L-arabinanase from Cellvibrio japonicus [11].


  1. Flipphi MJ, Visser J, van der Veen P, and de Graaff LH. Cloning of the Aspergillus niger gene encoding alpha-L-arabinofuranosidase A. Appl Microbiol Biotechnol. 1993 Jun;39(3):335-40. PubMed ID:7764056 | HubMed [Flipphi1993a]
  2. McKie VA, Black GW, Millward-Sadler SJ, Hazlewood GP, Laurie JI, and Gilbert HJ. Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action. Biochem J. 1997 Apr 15;323 ( Pt 2):547-55. PubMed ID:9163351 | HubMed [McKie1997]
  3. Flipphi MJ, Panneman H, van der Veen P, Visser J, and de Graaff LH. Molecular cloning, expression and structure of the endo-1,5-alpha-L-arabinase gene of Aspergillus niger. Appl Microbiol Biotechnol. 1993 Nov;40(2-3):318-26. PubMed ID:7764386 | HubMed [Flipphi1993b]
  4. Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, and Shoham Y. Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6. Biochemistry. 2005 Jan 11;44(1):387-97. DOI:10.1021/bi048059w | PubMed ID:15628881 | HubMed [Shallom2005]
  5. Ichinose H, Yoshida M, Kotake T, Kuno A, Igarashi K, Tsumuraya Y, Samejima M, Hirabayashi J, Kobayashi H, and Kaneko S. An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium. J Biol Chem. 2005 Jul 8;280(27):25820-9. DOI:10.1074/jbc.M501024200 | PubMed ID:15866877 | HubMed [Ichinose2005]
  6. Bourgois TM, Van Craeyveld V, Van Campenhout S, Courtin CM, Delcour JA, Robben J, and Volckaert G. Recombinant expression and characterization of XynD from Bacillus subtilis subsp. subtilis ATCC 6051: a GH 43 arabinoxylan arabinofuranohydrolase. Appl Microbiol Biotechnol. 2007 Jul;75(6):1309-17. DOI:10.1007/s00253-007-0956-2 | PubMed ID:17426966 | HubMed [Bourgois2007]
  7. van den Broek LA, Lloyd RM, Beldman G, Verdoes JC, McCleary BV, and Voragen AG. Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083. Appl Microbiol Biotechnol. 2005 Jun;67(5):641-7. DOI:10.1007/s00253-004-1850-9 | PubMed ID:15650848 | HubMed [vandenBroek2005]
  8. Matsuo N, Kaneko S, Kuno A, Kobayashi H, and Kusakabe I. Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901. Biochem J. 2000 Feb 15;346 Pt 1:9-15. PubMed ID:10657233 | HubMed [Matsuo2000]
  9. Pitson SM, Voragen AG, and Beldman G. Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases. FEBS Lett. 1996 Nov 25;398(1):7-11. PubMed ID:8946944 | HubMed [Pitson1996]
  10. Kersters-Hilderson H, Claeyssens M, van Doorslaer E, and de Bruyne CK. Determination of the anomeric configuration of D-xylose with D-xylose isomerases. Carbohydr Res. 1976 Apr;47(2):269-73. PubMed ID:1268883 | HubMed [KerstersHilderson1976]
  11. Nurizzo D, Turkenburg JP, Charnock SJ, Roberts SM, Dodson EJ, McKie VA, Taylor EJ, Gilbert HJ, and Davies GJ. Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold. Nat Struct Biol. 2002 Sep;9(9):665-8. DOI:10.1038/nsb835 | PubMed ID:12198486 | HubMed [Nurizzo2002]
  12. Jiang D, Fan J, Wang X, Zhao Y, Huang B, Liu J, and Zhang XC. Crystal structure of 1,3Gal43A, an exo-β-1,3-galactanase from Clostridium thermocellum. J Struct Biol. 2012 Dec;180(3):447-57. DOI:10.1016/j.jsb.2012.08.005 | PubMed ID:22960181 | HubMed [Jiang2012]
  13. Proctor MR, Taylor EJ, Nurizzo D, Turkenburg JP, Lloyd RM, Vardakou M, Davies GJ, and Gilbert HJ. Tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A. Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2697-702. DOI:10.1073/pnas.0500051102 | PubMed ID:15708971 | HubMed [Proctor2005]
  14. Vandermarliere E, Bourgois TM, Winn MD, van Campenhout S, Volckaert G, Delcour JA, Strelkov SV, Rabijns A, and Courtin CM. Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family. Biochem J. 2009 Feb 15;418(1):39-47. DOI:10.1042/BJ20081256 | PubMed ID:18980579 | HubMed [Vandermarliere2009]
  15. Brüx C, Ben-David A, Shallom-Shezifi D, Leon M, Niefind K, Shoham G, Shoham Y, and Schomburg D. The structure of an inverting GH43 beta-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues. J Mol Biol. 2006 May 26;359(1):97-109. DOI:10.1016/j.jmb.2006.03.005 | PubMed ID:16631196 | HubMed [Brux2006]
All Medline abstracts: PubMed | HubMed
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