Glycoside Hydrolase Family 16

• Author: Jens Eklöf
• Responsible Editor: 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 have evolved to exhibit predominantly xyloglucan 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 an 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 resque of the E134A mutant of a Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase resulting in an α-glycosyl product 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 family 16 three-dimensional structures have been solved of both 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 [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

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