Glycoside Hydrolase Family 158

This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.

• Author: ^^^Kazune Tamura^^^
• Responsible Curator: ^^^Harry Brumer^^^

---

Glycoside Hydrolase Family GH158
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH158.html

Substrate specificities

Members of family 158 have been shown to display activity towards β(1,3)-glucans, making it the fourth clan GH-A glycoside hydrolase family known to contain β(1,3)-glucanase activity, alongside GH17, GH128, and GH148. The founding member of this family, Vvad_PD1638 from Victivallis vadensis, was shown to be active on carboxymethyl-curdlan in a high-throughput screen [1].

BuGH158 from the human gut bacteria Bacteroides uniformis was the first GH158 member to receive detailed characterization [2]. BuGH158 is an endo β(1,3)-glucanase with high specificity towards laminarin from Laminaria digitata, a β(1,3)-glucan with single β(1,6)-glucose branches. BuGH158 is unable to tolerate more extensive branching as evidenced by poor activity towards other β(1,3)-glucans with longer, more frequent branches like laminarin from Eisenia bicyclis and yeast β-glucan [2]. The unbranched, linear β(1,3)-glucan curdlan was also not effectively hydrolyzed by BuGH158, due the glucans poor solubility in water (Vvad_PD1638 described above was active on a curdlan proxy that was chemically modified to increase water-solubility [1]).

Kinetics and Mechanism

As a family within clan GH-A, GH158 members were inferred to be retaining enzymes. Retention of anomeric stereochemistry was experimentally confirmed by ¹H NMR on the product of hydrolysis of 2-chloro-4-nitrophenyl laminaribioside by BuGH158 from Bacteroides uniformis [2]. As such, these enzymes follow the classical Koshland double-displacement mechanism, which proceeds via a covalent glycosyl-enzyme intermediate.

Catalytic Residues

The catalytic nucleophile and general acid/base residues of BuGH158 were predicted by structural homology with other clan GH-A members to be E220 and E137. The catalytic importance of these residues was subsequently confirmed by site-directed mutagenesis [2]. This glutamate pair is located on loops immediately following β-strands 7 (nucleophile) and 4 (acid/base), consistent with all other clan GH-A enzymes.

Three-dimensional structures

Figure 1. Structure of BuGH158. (PDB ID 6PAL) The TIM barrel domain is shown in cyan, the Ig-like domain in slate, and the catalytic nucleophile and acid/base glutamates are shown as sticks.

The X-ray crystal structure of BuGH158 from Bacteroides uniformis determined by multi-wavelength anomalous dispersion represents the founding structural representative of this family [2]. The 1.8 Å structure revealed a two-domain architecture with an N-terminal (α/β)₈ triose phosphate isomerase (TIM) barrel domain (which is a hallmark of clan GH-A structures) and a C-terminal eight-stranded immunoglobulin (Ig)-like domain that makes extensive contacts with the TIM barrel. A loop from the Ig-like domain extends over the TIM barrel to shape the active site cleft [2].

Family Firsts

First stereochemistry determination

Retention of product anomeric stereochemistry by BuGH158 from Bacteroides uniformis by ¹H NMR [2].

First catalytic nucleophile identification

E220 in BuGH158 from Bacteroides uniformis by structural study and kinetic analysis of site-directed mutant [2].

First general acid/base residue identification

E137 in BuGH158 from Bacteroides uniformis by structural study and kinetic analysis of site-directed mutant [2].

First 3-D structure

BuGH158 from Bacteroides uniformis by X-ray crystallography [2].

References

1. Helbert W, Poulet L, Drouillard S, Mathieu S, Loiodice M, Couturier M, Lombard V, Terrapon N, Turchetto J, Vincentelli R, and Henrissat B. (2019). Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space. Proc Natl Acad Sci U S A. 2019;116(13):6063-6068. DOI:10.1073/pnas.1815791116 |

2. Déjean G, Tamura K, Cabrera A, Jain N, Pudlo NA, Pereira G, Viborg AH, Van Petegem F, Martens EC, Brumer H. 2020. Synergy between cell-surface glycosidases and glycan-binding proteins dictates the utilization of specific beta(1,3)-glucans by human gut Bacteroides. mBio 11:e00095-20. DOI:10.1128/mBio.00095-20