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Glycoside Hydrolase Family 16

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

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 2.4.1.207, 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 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) [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 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].

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 [23]. 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. 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 | PubMed ID:27859885 | HubMed [McGregor2016]
  23. 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]
  24. 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
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