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Difference between revisions of "Glycoside Hydrolase Family 158"
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− | + | * [[Author]]: [[User:Kazune Tamura|Kazune Tamura]] | |
− | * [[Author]]: | + | * [[Responsible Curator]]: [[User:Harry Brumer|Harry Brumer]] |
− | * [[Responsible Curator]]: | ||
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== Substrate specificities == | == Substrate specificities == | ||
− | Members of family 158 have been shown to display activity towards β(1,3)-glucans, making | + | 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 <cite>Helbert2019</cite>. |
− | BuGH158 from the | + | BuGH158 from the human gut bacterium ''Bacteroides uniformis'' was the first GH158 member to receive detailed characterization <cite>Dejean2020</cite>. 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 <cite>Dejean2020</cite>. 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 <cite>Helbert2019</cite>). |
== Kinetics and Mechanism == | == 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 <sup>1</sup>H NMR on the product of hydrolysis of 2-chloro-4-nitrophenyl laminaribioside by BuGH158 | + | As a family within [[clan]] GH-A, GH158 members were inferred to be [[retaining]] enzymes. Retention of anomeric stereochemistry was experimentally confirmed by <sup>1</sup>H NMR on the product of hydrolysis of 2-chloro-4-nitrophenyl laminaribioside by BuGH158 <cite>Dejean2020</cite>. Thus, GH158 members enzymes employ the [[classical Koshland double-displacement mechanism]], which proceeds via a covalent glycosyl-enzyme [[intermediate]]. |
+ | |||
== Catalytic Residues == | == Catalytic Residues == | ||
− | The catalytic nucleophile and | + | 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 <cite>Dejean2020</cite>. 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 == | == Three-dimensional structures == | ||
− | The X-ray crystal structure of BuGH158 from Bacteroides uniformis determined by multi-wavelength anomalous dispersion represents the founding structural representative of this family | + | [[File:BuGH158.png|400px|thumb|right|'''Figure 1. Structure of BuGH158.''' ([{{PDBlink}}6PAL 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 <cite>Dejean2020</cite>. The 1.8 Å-resolution structure revealed a two-domain architecture with an N-terminal (α/β)<sub>8</sub> 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 <cite>Dejean2020</cite>. | ||
+ | |||
== Family Firsts == | == Family Firsts == | ||
− | ;First stereochemistry determination: | + | ;First stereochemistry determination: Retention of product anomeric stereochemistry by BuGH158 from ''Bacteroides uniformis'' using <sup>1</sup>H NMR <cite>Dejean2020</cite>. |
− | ;First catalytic nucleophile identification: | + | ;First [[catalytic nucleophile]] identification: E220 in BuGH158 from ''Bacteroides uniformis'' by tertiary structural homology and kinetic analysis of a site-directed mutant <cite>Dejean2020</cite>. |
− | ;First general acid/base residue identification: | + | ;First [[general acid/base]] residue identification: E137 in BuGH158 from ''Bacteroides uniformis'' by tertiary structural homology and kinetic analysis of a site-directed mutant <cite>Dejean2020</cite>. |
− | ;First 3-D structure: | + | ;First 3-D structure: BuGH158 from ''Bacteroides uniformis'' by X-ray crystallography <cite>Dejean2020</cite>. |
== References == | == References == | ||
<biblio> | <biblio> | ||
#Helbert2019 pmid=30850540 | #Helbert2019 pmid=30850540 | ||
− | # | + | #Dejean2020 pmid=32265336 |
</biblio> | </biblio> | ||
[[Category:Glycoside Hydrolase Families|GH158]] | [[Category:Glycoside Hydrolase Families|GH158]] |
Latest revision as of 14:15, 18 December 2021
This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.
Glycoside Hydrolase Family GH158 | |
Clan | GH-A |
Mechanism | retaining |
Active site residues | known |
CAZy DB link | |
http://www.cazy.org/GH158.html |
Substrate specificities
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 [1].
BuGH158 from the human gut bacterium 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 poor solubility of this polysaccharide in water (Vvad_PD1638 described above was active on a curdlan 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 [2]. Thus, GH158 members enzymes employ 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
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-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 [2].
Family Firsts
- First stereochemistry determination
- Retention of product anomeric stereochemistry by BuGH158 from Bacteroides uniformis using 1H NMR [2].
- First catalytic nucleophile identification
- E220 in BuGH158 from Bacteroides uniformis by tertiary structural homology and kinetic analysis of a site-directed mutant [2].
- First general acid/base residue identification
- E137 in BuGH158 from Bacteroides uniformis by tertiary structural homology and kinetic analysis of a site-directed mutant [2].
- First 3-D structure
- BuGH158 from Bacteroides uniformis by X-ray crystallography [2].
References
- 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 |
- 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. 2020;11(2). DOI:10.1128/mBio.00095-20 |