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• Author: Jens Eklöf and Antoni Planas
• Responsible Curator: Harry Brumer

Substrate specificities

Glycoside hydrolases of family 16 enzymes 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 2.4.1.207), keratan-sulfate endo-1,4-β-galactosidases (EC 3.2.1.103), endo-1,3-β-glucanases (EC 3.2.1.39), endo-1,3(4)-β-glucanases (EC 3.2.1.6), lichenases (EC 3.2.1.73), β-agarases (EC 3.2.1.81), κ-carrageenases (EC 3.2.1.83) and xyloglucanases (EC 3.2.1.151).

Kinetics and Mechanism

Family 16 enzymes are retaining enzymes, as first shown by NMR [2] 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 [3]. 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 [4]. 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 [4]. This mechanistic analysis on bacterial mixed-linkage endo-glucanases has been reviewed in the broader context of GH16 [5].

Three-dimensional structures

Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first solved 3D structure was a hybrid protein of lichenase M from Paenibacillus macerans and BglA from Bacillus amyloliquefaciens (PDB 1byh) in 1992 [6]. The first eukaryotic 3D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz) [7]. The first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) [8].

Evolution of GH16

Evolution of family 16 (click to enlarge)

Family 16 is a member of clan GH-B together with family 7 with whom they share their β-jellyroll fold. The different specificities of family 16 has been proposed to have evoloved from an ancestral β-1,3-glucanase [9]. 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 arisal of the lichenases and the XETs [10] (see figure).

Family firsts

First stereochemistry determination 

Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR [2].

First catalytic nucleophile identification 

Suggested in Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labelling[3]. Later verified in by azide rescue of inactivated mutants [4].

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 [11]. This was later verified by azide rescue of inactivated mutants [4].

First 3-D structure 

A hybrid lichenase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) [6].

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 pmid:17557806. PubMed HubMed [NXG]
2. 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. pmid:8280073. PubMed HubMed [REF3]
3. 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. pmid:1360982. PubMed HubMed [REF4]
4. 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 pmid:9698381. PubMed HubMed [7]
5. Planas A. Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering. Biochim Biophys Acta 2000 Dec 29; 1543(2) 361-382. pmid:11150614. PubMed HubMed [Planas2000]
6. 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. pmid:8099449. PubMed HubMed [5]
7. 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 pmid:15020748. PubMed HubMed [REF1]
8. 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 pmid:19154353. PubMed HubMed [8]
9. 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. pmid:9580981. PubMed HubMed [10]
10. 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. pmid:11435116. PubMed HubMed [9]
11. 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. pmid:8182059. PubMed HubMed [6]