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Glycoside Hydrolase Family 158
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|Glycoside Hydrolase Family GH158|
|Active site residues||known|
|CAZy DB link|
Members of family 158 have been shown to display activity towards β(1,3)-glucans, making this 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 .
BuGH158 from the human gut bacterium Bacteroides uniformis was the first GH158 member to receive detailed characterization . 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 . The unbranched, linear β(1,3)-glucan curdlan was also not effectively hydrolyzed by BuGH158, due the poor solubility of this polysaccharide in water (Vvad_PD1638 described above was active on a curdlan that was chemically modified to increase water-solubility ).
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 . Thus, GH158 members enzymes employ the classical Koshland double-displacement mechanism, which proceeds via a covalent glycosyl-enzyme intermediate.
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 . 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.
The X-ray crystal structure of BuGH158 from Bacteroides uniformis, determined by multi-wavelength anomalous dispersion, represents the founding structural representative of this family . The 1.8 Å-resolution structure revealed a two-domain architecture with an N-terminal (α/β)8 triose phosphate isomerase (TIM) barrel domain (the 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 .
- First stereochemistry determination
- Retention of product anomeric stereochemistry by BuGH158 from Bacteroides uniformis using 1H NMR .
- First catalytic nucleophile identification
- E220 in BuGH158 from Bacteroides uniformis by tertiary structural homology and kinetic analysis of a site-directed mutant .
- First general acid/base residue identification
- E137 in BuGH158 from Bacteroides uniformis by tertiary structural homology and kinetic analysis of a site-directed mutant .
- First 3-D structure
- BuGH158 from Bacteroides uniformis by X-ray crystallography .
- 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. 116, 6063-6068. DOI:10.1073/pnas.1815791116 |
- Déjean G, Tamura K, Cabrera A, Jain N, Pudlo NA, Pereira G, Viborg AH, Van Petegem F, Martens EC, and 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. DOI:10.1128/mBio.00095-20 |