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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

Substrate specificities

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 [1]. The substrate specificities found in GH16 are: xyloglucan:xyloglucosyltransferases (EC, keratan-sulfate endo-1,4-β-galactosidases (EC, endo-1,3-β-galactanases (EC 3.2.1.-), endo-1,3-β-glucanases (EC, endo-1,3(4)-β-glucanases (EC, lichenases (EC, β-agarases (EC, β-porphyranases (EC [2], κ-carrageenases (EC and xyloglucanases (EC

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 [3].

Kinetics and Mechanism

Family 16 enzymes are retaining enzymes, as first shown by NMR [4] 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 [5]. 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 [6]. 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 [6]. This mechanistic analysis on bacterial mixed-linkage endo-glucanases has been reviewed in the broader context of GH16 [7].

Three-dimensional structures

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 [8]. 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) [9]. The first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) [10].

Evolution of GH16

Evolution of family 16 (click to enlarge)

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 [11]. 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 [12] (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.

Family firsts

First stereochemistry determination 
Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR [4].
First catalytic nucleophile identification 
Suggested in Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labeling [5]. Later verified by azide rescue of inactivated mutants [6].
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 [13]. This was later verified by azide rescue of inactivated mutants [6].
First 3-D structure 
A hybrid lichenase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) [8].

Reference list

  1. 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]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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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