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Glycoside Hydrolase Family 16

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Glycoside Hydrolase Family 16
Clan GH-B
Mechanism retaining
Active site residues known
CAZy DB link
http://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:

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].

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

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  1. Hehemann JH, Correc G, Barbeyron T, Helbert W, Czjzek M, and Michel G. (2010) Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Nature. 464, 908-12. DOI:10.1038/nature08937 | PubMed ID:20376150 | HubMed [Hehemann2010]
  2. Eklöf JM and Brumer H. (2010) The XTH gene family: an update on enzyme structure, function, and phylogeny in xyloglucan remodeling. Plant Physiol. 153, 456-66. DOI:10.1104/pp.110.156844 | PubMed ID:20421457 | HubMed [Eklof2010]
  3. Error fetching PMID 18694928: [Cabib2008]
  4. Error fetching PMID 23919454: [Mazan2013]
  5. Error fetching PMID 25495733: [Blanco2015]
  6. Error fetching PMID 19712587: [Lee2009]
  7. Error fetching PMID 31501245: [Viborg2019]
  8. Error fetching PMID 8280073: [Malet1993]
  9. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006
    [Sinnott1990]
  10. Bissaro B, Monsan P, Fauré R, and O'Donohue MJ. (2015) Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases. Biochem J. 467, 17-35. DOI:10.1042/BJ20141412 | PubMed ID:25793417 | HubMed [Bissaro2015]
  11. Error fetching PMID 1360982: [Hoj1992]
  12. Error fetching PMID 9698381: [Viladot1998]
  13. Error fetching PMID 19419143: [Gullfot2009]
  14. Error fetching PMID 18043802: [Piens2007]
  15. Error fetching PMID 11150614: [Planas2000]
  16. Error fetching PMID 8099449: [Keitel1993]
  17. Error fetching PMID 15020748: [Johansson2004]
  18. Error fetching PMID 19154353: [Ilari2009]
  19. Error fetching PMID 9580981: [Barbeyron1998]
  20. Error fetching PMID 11435116: [Michel2001]
  21. Error fetching PMID 17557806: [Baumann2007]
  22. Error fetching PMID 23572521: [Eklof2013]
  23. Error fetching PMID 27859885: [McGregor2016]
  24. Error fetching PMID 29932263: [Behar2018]
  25. Error fetching PMID 8182059: [Juncosa1994]
  26. Error fetching PMID 21653698: [Kotake2011]
All Medline abstracts: PubMed | HubMed