Glycoside Hydrolase Family 16

• Author: Jens Eklöf
• Responsible Curator: Harry Brumer

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

Substrate specificities

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 nucleophile was first suggested 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-glycosyl substrate [4]. The acid-base was found 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].

Three-dimensional structures

Several three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin. The first solved 3-D structure was a hybrid protein of lichenase M from Paenibacillus macerans and BglA from Bacillus amyloliquefaciens (PDB 1byh) in 1992 [5]. The first eukaryotic 3-D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz) [6]. The first archeal 3-D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) [7].

Evolution of GH16

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 [8]. 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 [9] (see figure).

Evolution of family 16

Family firsts

First stereochemistry determination

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

First 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 [10]. 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) [5].

Reference list

1. Baumann MJ, Eklöf JM, Michel G, Kallas AM, Teeri TT, Czjzek M, and Brumer H 3rd. (2007). Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism. Plant Cell. 2007;19(6):1947-63. DOI:10.1105/tpc.107.051391 |
2. Malet C, Jiménez-Barbero J, Bernabé M, Brosa C, and Planas A. (1993). 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;296 ( Pt 3)(Pt 3):753-8. DOI:10.1042/bj2960753 |
3. Høj PB, Condron R, Traeger JC, McAuliffe JC, and Stone BA. (1992). 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;267(35):25059-66. | Google Books | Open Library
4. Viladot JL, de Ramon E, Durany O, and Planas A. (1998). 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;37(32):11332-42. DOI:10.1021/bi980586q |
5. Keitel T, Simon O, Borriss R, and Heinemann U. (1993). Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase. Proc Natl Acad Sci U S A. 1993;90(11):5287-91. DOI:10.1073/pnas.90.11.5287 |
6. Johansson P, Brumer H 3rd, Baumann MJ, Kallas AM, Henriksson H, Denman SE, Teeri TT, and Jones TA. (2004). Crystal structures of a poplar xyloglucan endotransglycosylase reveal details of transglycosylation acceptor binding. Plant Cell. 2004;16(4):874-86. DOI:10.1105/tpc.020065 |
7. Ilari A, Fiorillo A, Angelaccio S, Florio R, Chiaraluce R, van der Oost J, and Consalvi V. (2009). Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition. FEBS J. 2009;276(4):1048-58. DOI:10.1111/j.1742-4658.2008.06848.x |
8. Barbeyron T, Gerard A, Potin P, Henrissat B, and Kloareg B. (1998). The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases. Mol Biol Evol. 1998;15(5):528-37. DOI:10.1093/oxfordjournals.molbev.a025952 |
9. Michel G, Chantalat L, Duee E, Barbeyron T, Henrissat B, Kloareg B, and Dideberg O. (2001). 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;9(6):513-25. DOI:10.1016/s0969-2126(01)00612-8 |
10. Juncosa M, Pons J, Dot T, Querol E, and Planas A. (1994). 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;269(20):14530-5. | Google Books | Open Library