Glycoside Hydrolase Family 16
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|Glycoside Hydrolase Family 16|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolases of family 16 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 . The substrate specificities found in GH16 are: xyloglucan:xyloglucosyltransferases (EC 18.104.22.168), keratan-sulfate endo-1,4-β-galactosidases (EC 22.214.171.124), endo-1,3-β-galactanases (EC 3.2.1.-), endo-1,3-β-glucanases (EC 126.96.36.199), endo-1,3(4)-β-glucanases (EC 188.8.131.52), lichenases (EC 184.108.40.206), β-agarases (EC 220.127.116.11), β-porphyranases (EC 18.104.22.168) , κ-carrageenases (EC 22.214.171.124) and xyloglucanases (EC 126.96.36.199).
Some invertebrate GH16 proteins have lost their catalytic amino acids and are involved in immune response activation through the Toll pathway upon binding of β-1,3 glucan. The role of the GH16 domain in this immune response is still not elucidated .
Kinetics and Mechanism
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 . 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 . 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 . This mechanistic analysis on bacterial mixed-linkage endo-glucanases has been reviewed in the broader context of GH16 .
Proteins in family GH16 share the β-jelly-roll fold in which two β-sheets align in a sandwich like manner and its β-strands are bent around a perpendicular oriented substrate binding cleft. The first solved 3D structure was a hybrid protein of lichenase M from Paenibacillus macerans and BglA from Bacillus amyloliquefaciens (PDB 1byh) in 1992 . Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first eukaryotic 3D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz) . The first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) .
Evolution of GH16
Family 16 is a member of clan GH-B together with GH7 and both families share the β-jellyroll fold. The different specificities of family 16 were proposed to have been evolved from an ancestral β-1,3-glucanase . 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 lichenases and the XETs  (see figure). This evolutionary scenario was supported by a structure based phylogeny approach. In GH16 the active site residues are located in one beta-strand at the center of the substrate binding cleft and encoded within the signature motive EXDXXE or EXDXE. These motives feature two topologies, the beta-bulge motive which is more frequent in GH16 compared to the regular beta-strand, in which one amino acid is deleted. Due to the large expansion of the beta-bulge motive and its appearance in the related GH7 Michel et al. proposed that the ancestral enzyme of both families contained the beta-bulge explaining its wide distribution in GH16. This motive subsequently evolved to become the regular beta-strand that is common in contemporary XETs and lichenases.
- First stereochemistry determination
- Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR .
- First catalytic nucleophile identification
- Suggested in Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labeling . Later verified by azide rescue of inactivated mutants .
- 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 . This was later verified by azide rescue of inactivated mutants .
- First 3-D structure
- A hybrid lichenase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) .
- Baumann MJ, Eklöf JM, Michel G, Kallas AM, Teeri TT, Czjzek M, and Brumer H 3rd. Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism. Plant Cell. 2007 Jun;19(6):1947-63. DOI:10.1105/tpc.107.051391 |
- Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, and Michel G. Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature. 2010 Apr 8;464(7290):908-12. DOI:10.1038/nature08937 |
- Lee H, Kwon HM, Park JW, Kurokawa K, and Lee BL. 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 Aug 31;42(8):506-10.
- Malet C, Jiménez-Barbero J, Bernabé M, Brosa C, and Planas A. 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 Dec 15;296 ( Pt 3):753-8.
- Høj PB, Condron R, Traeger JC, McAuliffe JC, and Stone BA. 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 Dec 15;267(35):25059-66.
- Viladot JL, de Ramon E, Durany O, and Planas A. 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 Aug 11;37(32):11332-42. DOI:10.1021/bi980586q |
- Planas A. Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering. Biochim Biophys Acta. 2000 Dec 29;1543(2):361-382.
- Keitel T, Simon O, Borriss R, and Heinemann U. Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5287-91.
- Johansson P, Brumer H 3rd, Baumann MJ, Kallas AM, Henriksson H, Denman SE, Teeri TT, and Jones TA. Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding. Plant Cell. 2004 Apr;16(4):874-86. DOI:10.1105/tpc.020065 |
- Ilari A, Fiorillo A, Angelaccio S, Florio R, Chiaraluce R, van der Oost J, and Consalvi V. Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition. FEBS J. 2009 Feb;276(4):1048-58. DOI:10.1111/j.1742-4658.2008.06848.x |
- Barbeyron T, Gerard A, Potin P, Henrissat B, and Kloareg B. The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases. Mol Biol Evol. 1998 May;15(5):528-37.
- Michel G, Chantalat L, Duee E, Barbeyron T, Henrissat B, Kloareg B, and Dideberg O. 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 Jun;9(6):513-25.
- Juncosa M, Pons J, Dot T, Querol E, and Planas A. 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 May 20;269(20):14530-5.
- Kotake T, Hirata N, Degi Y, Ishiguro M, Kitazawa K, Takata R, Ichinose H, Kaneko S, Igarashi K, Samejima M, and Tsumuraya Y. Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes. J Biol Chem. 2011 Aug 5;286(31):27848-54. DOI:10.1074/jbc.M111.251736 |