Glycoside Hydrolase Family 16

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Author: Jens Eklöf and ^^^Jan-Hendrik Hehemann^^^
Responsible Curator: ^^^Harry Brumer^^^

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

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 2.4.1.207), keratan-sulfate endo-1,4-β-galactosidases (EC 3.2.1.103), endo-1,3-β-galactanases (EC 3.2.1.-), 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), β-porphyranases (EC 3.2.1.178) [2], κ-carrageenases (EC 3.2.1.83) and xyloglucanases (EC 3.2.1.151).

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].

Polysaccharides cleaved by GH16 enzymes
Agar
β-D-Galp-(1,4)-α-L-3,6-anhydro-Galp-1,3-β-D-Galp-(1,4)-α-L-3,6-anhydro-Galp
κ-carrageenan
β-D-Galp-(1,4)-α-D-3,6-anhydro-Galp-1,3-β-D-Galp-(1,4)-α-D-3,6-anhydro-Galp
Porphyran
β-1,3-glucan
β-D-Glcp-(1,3)- β-D-Glcp-(1,3)- β-D-Glcp-(1,3)- β-D-Glcp
Keratan sulphate
β-D-Galp-(1,4)- β-D-GlcpNAc 6-sulphate-(1,3)- β-D-Galp-(1,4)- β-D-GlcpNAc 6-sulphate
β-1,3-galactan
β-D-Galp-(1,3)- β-D-Galp-(1,3)- β-D-Galp-(1,3)- β-D-Galp
β-1,4/1,3-glucan
β-D-Glcp-(1,4)- β-D-Glcp-(1,4)- β-D-Glcp-(1,3)- β-D-Glcp-(1,4)- β-D-Glcp
β-1,4-galactan
β-D-Galp-(1,4)- β-D-Galp-(1,4)- β-D-Galp-(1,4)- β-D-Galp
Xyloglucan

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

All Medline abstracts: PubMed