Glycoside Hydrolase Family 16
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|Glycoside Hydrolase Family 16|
|Active site residues||known|
|CAZy DB link|
The members of family 16 are active on β-1,4 or β-1,3 glycosidic bonds in various glucans and galactans. A wide diversity of glycoside hydrolases active on plant and marine polysaccharides are found in GH16, including:
- keratan-sulfate endo-1,4-β-galactosidases (EC 220.127.116.11),
- endo-1,3-β-galactanases (EC 3.2.1.-),
- endo-1,3-β-glucanases (EC 18.104.22.168),
- endo-1,3(4)-β-glucanases (EC 22.214.171.124),
- licheninases (EC 126.96.36.199),
- β-agarases (EC 188.8.131.52),
- β-porphyranases (EC 184.108.40.206) ,
- κ-carrageenases (EC 220.127.116.11), and
- endo-xyloglucanases (EC 18.104.22.168, a.k.a. xyloglucan endo-hydrolases, XEHs, in plants ).
Notably, some members of GH16 are predominant transglycosylases. These include the plant xyloglucan:xyloglucosyltransferases (EC 22.214.171.124, a.k.a. xyloglucan endo-transglycosylases, XETs)  and yeast chitin/beta-glucan crosslinking enzymes Crh1 and Crh2 [3, 4, 5].
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 has not been fully elucidated .
Kinetics and Mechanism
Members of GH16 enzymes are retaining enzymes, as first shown by NMR  on an endo-1,3-1,4-β-D-glucan 4-glucanohydrolase from Bacillus licheniformis. As such, they utilize a covalent glycosyl-enzyme intermediate, which is broken-down by glycosyl transfer [8, 9] to water or a carbohydrate acceptor substrate in glycoside hydrolases or transglycosylases, respectively.
The catalytic nucleophile of GH16 enzymes was first proposed using a non-specific epoxyalkyl β-glycoside inhibitor and identification of the site of covalent labelling using 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 site-directed mutant of the Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase together with kinetic analysis and azide rescue, which resulted in a β-glycosyl azide product . These structurally conserved catalytic residues have been confirmed in a number of other GH16 members, including plant XETs and XEHs [12, 13], and yeast Crh1 and Crh2 .
Proteins in GH16 share a β-jelly-roll fold in which two β-sheets align in a curved, sandwich-like manner and present a cleft-shaped active-site bounded by loops extending from the β-strands. The first solved 3D structure was a hybrid protein of licheninase M from Paenibacillus macerans and BglA from Bacillus amyloliquefaciens (PDB 1byh) in 1992 . Many three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin (see http://www.cazy.org/GH16_structure.html for an updated list). Of these, the first eukaryotic 3D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz)  and the first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) .
Evolution of GH16
GH16 is a member of clan GH-B together with GH7; both families share the β-jellyroll fold. The different specificities of GH16 are proposed to have evolved from an ancestral β-1,3-glucanase . This proposal was elaborated using a structure-based phylogeny approach, which suggested that a first branching event lead to the evolution of the bacterial κ-carrageenases and the β-agarases, while a later branching event lead to the bacterial licheninases and the plant XETs  (Figure 1). In particular, the GH16 active-site residues are located in-train on one beta-strand at the center of the substrate binding cleft. Depending upon the phylogenetic clade, this beta-strand features one of two topologies. The beta-bulge motif, which has the consensus sequence EXDXXE, is more frequent in GH16 compared to the regular beta-strand with the consensus sequence EXDXE (the catalytic nucleophile is the first glutamate and the catalytic acid/base is the second, with a proposed "helper" asparate in-between ). Due to the predominance of the beta-bulge motif and its presence as the only motif in GH7, Michel et al. proposed that the beta-bulge is the ancestral motif, which subsequently gave rise to the regular beta-strand of extant XETs and licheninases . More recently, similar structure-based phylogenetic approaches have suggested that XEHs evolved subsequently to XEHs within the xyloglucan endo-transglycosylase/hydrolase (XTH) gene family in plant lineages [2, 20], while the more recent identification of a group of bifunctional GH16 glycoside hydrolases, which is active on both mixed-linkage beta-glucan and xyloglucan, provides additional support for the close evolutionary relationship of XETs and licheninases [21, 22].
- 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 licheninase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) .
- 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 |
- Eklöf JM and Brumer H. The XTH gene family: an update on enzyme structure, function, and phylogeny in xyloglucan remodeling. Plant Physiol. 2010 Jun;153(2):456-66. DOI:10.1104/pp.110.156844 |
- Cabib E, Farkas V, Kosík O, Blanco N, Arroyo J, and McPhie P. Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro. J Biol Chem. 2008 Oct 31;283(44):29859-72. DOI:10.1074/jbc.M804274200 |
- Mazáň M, Blanco N, Kováčová K, Firáková Z, Rehulka P, Farkaš V, and Arroyo J. A novel fluorescence assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases. Biochem J. 2013 Nov 1;455(3):307-18. DOI:10.1042/BJ20130354 |
- Blanco N, Sanz AB, Rodríguez-Peña JM, Nombela C, Farkaš V, Hurtado-Guerrero R, and Arroyo J. Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases. FEBS J. 2015 Feb;282(4):715-31. DOI:10.1111/febs.13176 |
- 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.
- Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006
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- 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 |
- Gullfot F, Ibatullin FM, Sundqvist G, Davies GJ, and Brumer H. Functional characterization of xyloglucan glycosynthases from GH7, GH12, and GH16 scaffolds. Biomacromolecules. 2009 Jul 13;10(7):1782-8. DOI:10.1021/bm900215p |
- Piens K, Henriksson AM, Gullfot F, Lopez M, Fauré R, Ibatullin FM, Teeri TT, Driguez H, and Brumer H. Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants. Org Biomol Chem. 2007 Dec 21;5(24):3971-8. DOI:10.1039/b714570e |
- 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.
- 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 |
- Eklöf JM, Shojania S, Okon M, McIntosh LP, and Brumer H. Structure-function analysis of a broad specificity Populus trichocarpa endo-β-glucanase reveals an evolutionary link between bacterial licheninases and plant XTH gene products. J Biol Chem. 2013 May 31;288(22):15786-99. DOI:10.1074/jbc.M113.462887 |
- McGregor N, Yin V, Tung CC, Van Petegem F, and Brumer H. Crystallographic insight into the evolutionary origins of xyloglucan endo-transglycosylases and endo-hydrolases. Plant J. 2016 Nov 15. DOI:10.1111/tpj.13421 |
- 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 |