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
https://www.cazy.org/GH16.html

Substrate specificities

The members of family 16 are active on β-1,4 or β-1,3 glycosidic bonds in various glucans and galactans. A wide diversity of glycoside hydrolases active on plant and marine polysaccharides are found in GH16, including:

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),
licheninases (EC 3.2.1.73),
β-agarases (EC 3.2.1.81),
β-porphyranases (EC 3.2.1.178) [1],
κ-carrageenases (EC 3.2.1.83), and
endo-xyloglucanases (EC 3.2.1.151, a.k.a. xyloglucan endo-hydrolases, XEHs, in plants [2]).

Notably, some members of GH16 are predominant transglycosylases. These include the plant xyloglucan:xyloglucosyltransferases (EC 2.4.1.207, a.k.a. xyloglucan endo-transglycosylases, XETs) [2] and yeast chitin/beta-glucan crosslinking enzymes Crh1 and Crh2 [3, 4, 5]. 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 has not been fully elucidated [6].

Several of the activities observed for GH16 members are delineated into individual sequence-based subfamilies, while other polyspecific subfamilies capture a range of activities [7].

Diverse 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

Members of GH16 enzymes are retaining enzymes, as first shown by NMR [8] on an endo-1,3-1,4-β-D-glucan 4-glucanohydrolase from Bacillus licheniformis. As such, they utilize a covalent glycosyl-enzyme intermediate, which is broken-down by glycosyl transfer [9, 10] to water or a carbohydrate acceptor substrate in glycoside hydrolases or transglycosylases, respectively.

Catalytic Residues

The catalytic nucleophile of GH16 enzymes was first proposed using a non-specific epoxyalkyl β-glycoside inhibitor and identification of the site of covalent labelling using ESI-MS and Edman degradation on an endo-1,3-1,4-β-D-glucan 4-glucanohydrolase from Bacillus amyloliquefaciens [11]. 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 [12]. The general acid/base residue was identified by making the E138A site-directed mutant of the Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase together with kinetic analysis and azide rescue, which resulted in a β-glycosyl azide product [12]. These structurally conserved catalytic residues have been confirmed in a number of other GH16 members, including plant XETs and XEHs [13, 14], and yeast Crh1 and Crh2 [5].

The mechanistic analysis of bacterial mixed-linkage endo-glucanases has been expertly reviewed in the broader context of GH16 [15].

Three-dimensional structures

Proteins in GH16 share a β-jelly-roll fold in which two β-sheets align in a curved, sandwich-like manner and present a cleft-shaped active-site bounded by loops extending from the β-strands. The first solved 3D structure was a hybrid protein of licheninase M from Paenibacillus macerans and BglA from Bacillus amyloliquefaciens (PDB 1byh) in 1992 [16]. Many three-dimensional structures have been solved of family 16 members of archeal, bacterial, and eukaryotic origin (see http://www.cazy.org/GH16_structure.html for an updated list). Of these, the first eukaryotic 3D structure was the xyloglucan endo-transglycosylase PttXET16-34 from Populus tremula×tremuloides (PDB 1umz) [17] and the first archeal 3D structure was a endo-1,3-β-glucanase Lam16 from Pyrococcus furiosus (PDB 2vy0) [18].

The structural diversity of GH16 members across sequence-related subfamilies has been reviewed in detail [7].

Evolution of GH16

Figure 1. Proposed evolution of GH16 (click to enlarge).

GH16 is a member of clan GH-B together with GH7; both families share the β-jellyroll fold. The different specificities of GH16 are proposed to have evolved from an ancestral β-1,3-glucanase [19]. This proposal was first elaborated using a structure-based phylogeny approach, which suggested that an early branching event lead to the evolution of the bacterial κ-carrageenases and the β-agarases, while a later branching event lead to the bacterial licheninases and the plant XETs [20] (Figure 1). GH16 has more recently been divided into subfamilies within CAZy, the corresponding phylogenetic analysis of which supports this overall evolutionary trajectory [7].

Particularly notable, the GH16 active-site residues are located in-train on one beta-strand at the center of the substrate binding cleft. Depending upon the phylogenetic clade, this beta-strand features one of two topologies. The beta-bulge motif, which has the consensus sequence EXDXXE, is more frequent in GH16 compared to the regular beta-strand with the consensus sequence EXDXE (the catalytic nucleophile is the first glutamate and the catalytic acid/base is the second, with a proposed "helper" asparate in-between [15]). Due to the predominance of the beta-bulge motif and its presence as the only motif in GH7, Michel et al. proposed that the beta-bulge is the ancestral motif, which subsequently gave rise to the regular beta-strand of extant plant XETs and bacterial licheninases [20].

Within plant lineages, similar structure-based phylogenetic approaches have suggested that XEHs evolved subsequently to XEHs within the xyloglucan endo-transglycosylase/hydrolase (XTH) gene family [2, 21]. The identification of a group of bifunctional GH16 glycoside hydrolases, which is active on both mixed-linkage beta-glucan and xyloglucan, provides additional support for the close evolutionary relationship of XETs and licheninases [22, 23, 24].

Family firsts

First stereochemistry determination: Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by NMR [8].
First catalytic nucleophile identification: Suggested in Bacillus amyloliquefaciens 1,3-1,4-β-D-glucan 4-glucanohydrolase via non-specific epoxyalkyl β-glycoside labeling [11]. Later verified by azide rescue of inactivated mutants [12].
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 [25]. This was later verified by azide rescue of inactivated mutants [12].
First 3-D structure: A hybrid licheninase (Bacillus amyloliquefaciens and Paenibacillus macerans) by X-ray crystallography (PDB 1byh) [16].

Reference list

All Medline abstracts: PubMed