Glycoside Hydrolase Family 16

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

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

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

All Medline abstracts: PubMed