Glycoside Hydrolase Family 16

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• Author: Jens Eklöf and ^^^Jan-Hendrik Hehemann^^^
• Responsible Curator: ^^^Harry Brumer^^^

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Glycoside Hydrolase Family 16
Clan	GH-B
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH16.html

Substrate specificities

Glycoside hydrolases of family 16 enzymes cleave β-1,4 or β-1,3 glycosidic bonds in various glucans and galactans. Some members of this family operating on xyloglucan exhibit predominant endo-transglycosylase activity [1]. The substrate specificities found in GH16 are: xyloglucan:xyloglucosyltransferases (EC 2.4.1.207), keratan-sulfate endo-1,4-β-galactosidases (EC 3.2.1.103), endo-1,3-β-galactanases (EC 3.2.1.-), endo-1,3-β-glucanases (EC 3.2.1.39), endo-1,3(4)-β-glucanases (EC 3.2.1.6), lichenases (EC 3.2.1.73), β-agarases (EC 3.2.1.81), β-porphyranases (EC 3.2.1.178) [2], κ-carrageenases (EC 3.2.1.83) and xyloglucanases (EC 3.2.1.151).

Some invertebrate GH16 proteins have lost their catalytic amino acids and are involved in immune response activation through the Toll pathway upon binding of fungal β 1,3 glucan. The role of the GH16 domain in this immune response is still not elucidated [3, 4, 4, 5, 6, 7, 8, 9, 10, 10, 11, 12, 13, 14, 15, 16, 17, 18] on an endo-1,3-1,4-β-D-glucan 4-glucanohydrolase from Bacillus licheniformis.

Catalytic Residues

The catalytic nucleophile was first proposed using a non-specific epoxyalkyl β-glycoside inhibitor and subsequent peptide identification by ESI-MS and Edman degradation on an endo-1,3-1,4-β-D-glucan 4-glucanohydrolase from Bacillus amyloliquefaciens [19]. This was subsequently verified by azide rescue of the E134A mutant of a Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase resulting in an α-glycosyl azide from the β-glycoside substrate [20]. The general acid/base residue was identified by making the E138A mutant from the Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase and subsequent azide rescue resulting in a β-glycosyl azide product [20]. This mechanistic analysis on bacterial mixed-linkage endo-glucanases has been reviewed in the broader context of GH16 [21].

Three-dimensional structures

Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first solved 3D structure was a hybrid protein of lichenase M from Paenibacillus macerans and BglA from Bacillus amyloliquefaciens (PDB 1byh) in 1992 [22]. The first eukaryotic 3D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz) [23]. The first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) [24].

Evolution of GH16

Evolution of family 16 (click to enlarge)

Family 16 is a member of clan GH-B together with family 7 with whom they share their β-jellyroll fold. The different specificities of family 16 has been proposed to have evolved from an ancestral β-1,3-glucanase [25]. The first branching in family 16 lead to the evolution of the κ-carrageenases and the β-agarases and a later branching event lead to the arisal of the lichenases and the XETs [26] (see figure).

Family firsts

First stereochemistry determination

Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR [18].

First catalytic nucleophile identification

Suggested in Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labelling[19]. Later verified in by azide rescue of inactivated mutants [20].

First general acid/base residue identification

Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase, first suggested by sequence homology and mutational studies [27]. This was later verified by azide rescue of inactivated mutants [20].

First 3-D structure

A hybrid lichenase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) [22].

Reference list

1. Baumann MJ, Eklöf JM, Michel G, Kallas AM, Teeri TT, Czjzek M, and Brumer H 3rd. (2007). Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism. Plant Cell. 2007;19(6):1947-63. DOI:10.1105/tpc.107.051391 |
2. Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, and Michel G. (2010). Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature. 2010;464(7290):908-12. DOI:10.1038/nature08937 |
3. Lee H, Kwon HM, Park JW, Kurokawa K, and Lee BL. (2009). N-terminal GNBP homology domain of Gram-negative binding protein 3 functions as a beta-1,3-glucan binding motif in Tenebrio molitor. BMB Rep. 2009;42(8):506-10. DOI:10.5483/bmbrep.2009.42.8.506 |
18. Malet C, Jiménez-Barbero J, Bernabé M, Brosa C, and Planas A. (1993). Stereochemical course and structure of the products of the enzymic action of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis. Biochem J. 1993;296 ( Pt 3)(Pt 3):753-8. DOI:10.1042/bj2960753 |
19. Høj PB, Condron R, Traeger JC, McAuliffe JC, and Stone BA. (1992). Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors. J Biol Chem. 1992;267(35):25059-66. | Google Books | Open Library
20. Viladot JL, de Ramon E, Durany O, and Planas A. (1998). Probing the mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues. Biochemistry. 1998;37(32):11332-42. DOI:10.1021/bi980586q |
21. Planas A (2000). Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering. Biochim Biophys Acta. 2000;1543(2):361-382. DOI:10.1016/s0167-4838(00)00231-4 |
22. Keitel T, Simon O, Borriss R, and Heinemann U. (1993). Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase. Proc Natl Acad Sci U S A. 1993;90(11):5287-91. DOI:10.1073/pnas.90.11.5287 |
23. Johansson P, Brumer H 3rd, Baumann MJ, Kallas AM, Henriksson H, Denman SE, Teeri TT, and Jones TA. (2004). Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding. Plant Cell. 2004;16(4):874-86. DOI:10.1105/tpc.020065 |
24. Ilari A, Fiorillo A, Angelaccio S, Florio R, Chiaraluce R, van der Oost J, and Consalvi V. (2009). Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition. FEBS J. 2009;276(4):1048-58. DOI:10.1111/j.1742-4658.2008.06848.x |
25. Barbeyron T, Gerard A, Potin P, Henrissat B, and Kloareg B. (1998). The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases. Mol Biol Evol. 1998;15(5):528-37. DOI:10.1093/oxfordjournals.molbev.a025952 |
26. Michel G, Chantalat L, Duee E, Barbeyron T, Henrissat B, Kloareg B, and Dideberg O. (2001). The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases. Structure. 2001;9(6):513-25. DOI:10.1016/s0969-2126(01)00612-8 |
27. Juncosa M, Pons J, Dot T, Querol E, and Planas A. (1994). Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis. J Biol Chem. 1994;269(20):14530-5. | Google Books | Open Library
28. Kotake T, Hirata N, Degi Y, Ishiguro M, Kitazawa K, Takata R, Ichinose H, Kaneko S, Igarashi K, Samejima M, and Tsumuraya Y. (2011). Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes. J Biol Chem. 2011;286(31):27848-54. DOI:10.1074/jbc.M111.251736 |