New to the CAZy classification? Read this first.

Glycoside Hydrolase Family 16

From CAZypedia
Jump to: navigation, search
Approve icon-50px.png
This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail, or using this form.

Glycoside Hydrolase Family 16
Clan GH-B
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

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:

Notably, some members of GH16 are predominant transglycosylases. These include the plant xyloglucan:xyloglucosyltransferases (EC, a.k.a. xyloglucan endo-transglycosylases, XETs) [2] 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 [6].

Kinetics and Mechanism

Members of GH16 enzymes are retaining enzymes, as first shown by NMR [7] 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.

Catalytic Residues

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 [10]. 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 [11]. 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 [11]. 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 [5].

The mechanistic analysis of bacterial mixed-linkage endo-glucanases has been expertly reviewed in the broader context of GH16 [14].

Three-dimensional structures

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 [15]. Many three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin (see for an updated list). Of these, the first eukaryotic 3D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz) [16] and the first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) [17].

Evolution of GH16

Figure 1. Proposed evolution of GH16 (click to enlarge).

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 [18]. 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 [19] (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 [14]). 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 [19]. 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 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].

Family firsts

First stereochemistry determination 
Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR [7].
First catalytic nucleophile identification 
Suggested in Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labeling [10]. Later verified by azide rescue of inactivated mutants [11].
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 [22]. This was later verified by azide rescue of inactivated mutants [11].
First 3-D structure 
A hybrid licheninase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) [15].

Reference list

  1. 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 | PubMed ID:20376150 | HubMed [Hehemann2010]
  2. 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 | PubMed ID:20421457 | HubMed [Eklof2010]
  3. 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 | PubMed ID:18694928 | HubMed [Cabib2008]
  4. 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 | PubMed ID:23919454 | HubMed [Mazan2013]
  5. 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 | PubMed ID:25495733 | HubMed [Blanco2015]
  6. 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. PubMed ID:19712587 | HubMed [Lee2009]
  7. 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. PubMed ID:8280073 | HubMed [Malet1993]
  8. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006 [Sinnott1990]
  9. Bissaro B, Monsan P, Fauré R, and O'Donohue MJ. Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases. Biochem J. 2015 Apr 1;467(1):17-35. DOI:10.1042/BJ20141412 | PubMed ID:25793417 | HubMed [Bissaro2015]
  10. 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. PubMed ID:1360982 | HubMed [Hoj1992]
  11. 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 | PubMed ID:9698381 | HubMed [Viladot1998]
  12. 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 | PubMed ID:19419143 | HubMed [Gullfot2009]
  13. 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 | PubMed ID:18043802 | HubMed [Piens2007]
  14. Planas A. Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering. Biochim Biophys Acta. 2000 Dec 29;1543(2):361-382. PubMed ID:11150614 | HubMed [Planas2000]
  15. 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. PubMed ID:8099449 | HubMed [Keitel1993]
  16. 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 | PubMed ID:15020748 | HubMed [Johansson2004]
  17. 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 | PubMed ID:19154353 | HubMed [Ilari2009]
  18. 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. PubMed ID:9580981 | HubMed [Barbeyron1998]
  19. 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. PubMed ID:11435116 | HubMed [Michel2001]
  20. 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 | PubMed ID:17557806 | HubMed [Baumann2007]
  21. 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 | PubMed ID:23572521 | HubMed [Eklof2013]
  22. 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. PubMed ID:8182059 | HubMed [Juncosa1994]
  23. 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 | PubMed ID:21653698 | HubMed [Kotake2011]
All Medline abstracts: PubMed | HubMed
Personal tools

About CAZypedia
For contributors