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Difference between revisions of "Glycoside Hydrolase Family 158"

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== Substrate specificities ==
 
== Substrate specificities ==
Members of family 158 have been shown to display activity towards β(1,3)-glucans, making it the fourth clan GH-A 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>.
+
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 <cite>Helbert2019</cite>.
  
BuGH158 from the prominent human gut symbiont ''Bacteroides uniformis'' was the first GH158 member to receive detailed characterization <cite>Dejean2019</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>Dejean2019</cite>. 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 <cite>Helbert2019</cite>).
+
BuGH158 from the human gut bacteria ''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 glucans poor solubility in water (Vvad_PD1638 described above was active on a curdlan proxy 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 from Bacteroides uniformis <cite>Dejean2019</cite>. As such, these enzymes follow the classical Koshland double-displacement mechanism, which proceed via a covalent glycosyl-enzyme intermediate.
+
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 from ''Bacteroides uniformis'' <cite>Dejean2020</cite>. As such, these enzymes follow the [[classical Koshland double-displacement mechanism]], which proceed via a covalent glycosyl-enzyme [[intermediate]].
 
== Catalytic Residues ==
 
== Catalytic Residues ==
The catalytic nucleophile and catalytic acid/base residues of BuGH158 were determined to be E220 and E137 <cite>Dejean2019</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.
+
The [[catalytic nucleophile]] and [[general acid/base]] residues of BuGH158 were determined to be E220 and E137 <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 ==
[[File:BuGH158.png]]
+
[[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>Dejean2019</cite>. The 1.8 Å structure revealed in addition to an N-terminal (α/β)<sub>8</sub> triose phosphate isomerase (TIM) barrel domain, which is a hallmark of clan GH-A structures, 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>Dejean2019</cite>.
+
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 structure revealed a two-domain architecture with an N-terminal (α/β)<sub>8</sub> 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 <cite>Dejean2020</cite>.
 
== Family Firsts ==
 
== Family Firsts ==
;First stereochemistry determination: Retention of product anomeric stereochemistry by BuGH158 from ''Bacteroides uniformis'' by NMR <cite>Dejean2019</cite>.
+
;First stereochemistry determination: Retention of product anomeric stereochemistry by BuGH158 from ''Bacteroides uniformis'' by <sup>1</sup>H NMR <cite>Dejean2020</cite>.
;First catalytic nucleophile identification: E220 in BuGH158 from ''Bacteroides uniformis'' by structural study and kinetic analysis of site-directed mutant <cite>Dejean2019</cite>.
+
;First [[catalytic nucleophile]] identification: E220 in BuGH158 from ''Bacteroides uniformis'' by structural study and kinetic analysis of site-directed mutant <cite>Dejean2020</cite>.
;First general acid/base residue identification: E137 in BuGH158 from ''Bacteroides uniformis'' by structural study and kinetic analysis of site-directed mutant <cite>Dejean2019</cite>.
+
;First [[general acid/base]] residue identification: E137 in BuGH158 from ''Bacteroides uniformis'' by structural study and kinetic analysis of site-directed mutant <cite>Dejean2020</cite>.
;First 3-D structure: BuGH158 from ''Bacteroides uniformis'' by X-ray crystallography <cite>Dejean2019</cite>.
+
;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
#Dejean2019 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. https://doi.org/10.1128/ mBio.00095-20.  
+
#Dejean2020 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. https://doi.org/10.1128/ mBio.00095-20.  
 
</biblio>
 
</biblio>
  
 
[[Category:Glycoside Hydrolase Families|GH158]]
 
[[Category:Glycoside Hydrolase Families|GH158]]

Revision as of 16:00, 20 March 2020

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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 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 proceed via a covalent glycosyl-enzyme intermediate.

Catalytic Residues

The catalytic nucleophile and general acid/base residues of BuGH158 were determined to be E220 and E137 [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. 116, 6063-6068. DOI:10.1073/pnas.1815791116 | PubMed ID:30850540 | HubMed [Helbert2019]
  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. https://doi.org/10.1128/ mBio.00095-20.
    [Dejean2020]