Glycoside Hydrolase Family 43

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• Author: Harry Gilbert
• Responsible Curator: Harry Gilbert

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

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 pK_a 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 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 [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].

References

1. Flipphi MJ, Visser J, van der Veen P, and de Graaff LH. (1993). Cloning of the Aspergillus niger gene encoding alpha-L-arabinofuranosidase A. Appl Microbiol Biotechnol. 1993;39(3):335-40. DOI:10.1007/BF00192088 |
2. McKie VA, Black GW, Millward-Sadler SJ, Hazlewood GP, Laurie JI, and Gilbert HJ. (1997). Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action. Biochem J. 1997;323 ( Pt 2)(Pt 2):547-55. DOI:10.1042/bj3230547 |
3. Flipphi MJ, Panneman H, van der Veen P, Visser J, and de Graaff LH. (1993). Molecular cloning, expression and structure of the endo-1,5-alpha-L-arabinase gene of Aspergillus niger. Appl Microbiol Biotechnol. 1993;40(2-3):318-26. DOI:10.1007/BF00170387 |
4. Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, and Shoham Y. (2005). Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6. Biochemistry. 2005;44(1):387-97. DOI:10.1021/bi048059w |
5. Ichinose H, Yoshida M, Kotake T, Kuno A, Igarashi K, Tsumuraya Y, Samejima M, Hirabayashi J, Kobayashi H, and Kaneko S. (2005). An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium. J Biol Chem. 2005;280(27):25820-9. DOI:10.1074/jbc.M501024200 |
6. Bourgois TM, Van Craeyveld V, Van Campenhout S, Courtin CM, Delcour JA, Robben J, and Volckaert G. (2007). Recombinant expression and characterization of XynD from Bacillus subtilis subsp. subtilis ATCC 6051: a GH 43 arabinoxylan arabinofuranohydrolase. Appl Microbiol Biotechnol. 2007;75(6):1309-17. DOI:10.1007/s00253-007-0956-2 |
7. van den Broek LA, Lloyd RM, Beldman G, Verdoes JC, McCleary BV, and Voragen AG. (2005). Cloning and characterization of arabinoxylan arabinofuranohydrolase-D3 (AXHd3) from Bifidobacterium adolescentis DSM20083. Appl Microbiol Biotechnol. 2005;67(5):641-7. DOI:10.1007/s00253-004-1850-9 |
8. Matsuo N, Kaneko S, Kuno A, Kobayashi H, and Kusakabe I. (2000). Purification, characterization and gene cloning of two alpha-L-arabinofuranosidases from streptomyces chartreusis GS901. Biochem J. 2000;346 Pt 1(Pt 1):9-15. | Google Books | Open Library
9. 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 |
10. Kersters-Hilderson H, Claeyssens M, van Doorslaer E, and de Bruyne CK. (1976). Determination of the anomeric configuration of D-xylose with D-xylose isomerases. Carbohydr Res. 1976;47(2):269-73. DOI:10.1016/s0008-6215(00)84192-0 |
11. 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 |
12. Jiang D, Fan J, Wang X, Zhao Y, Huang B, Liu J, and Zhang XC. (2012). Crystal structure of 1,3Gal43A, an exo-β-1,3-galactanase from Clostridium thermocellum. J Struct Biol. 2012;180(3):447-57. DOI:10.1016/j.jsb.2012.08.005 |
13. Proctor MR, Taylor EJ, Nurizzo D, Turkenburg JP, Lloyd RM, Vardakou M, Davies GJ, and Gilbert HJ. (2005). 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;102(8):2697-702. DOI:10.1073/pnas.0500051102 |
14. Vandermarliere E, Bourgois TM, Winn MD, van Campenhout S, Volckaert G, Delcour JA, Strelkov SV, Rabijns A, and Courtin CM. (2009). 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;418(1):39-47. DOI:10.1042/BJ20081256 |
15. Brüx C, Ben-David A, Shallom-Shezifi D, Leon M, Niefind K, Shoham G, Shoham Y, and Schomburg D. (2006). 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;359(1):97-109. DOI:10.1016/j.jmb.2006.03.005 |