Difference between revisions of "Glycoside Hydrolase Family 162"

Revision as of 23:37, 3 June 2019

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Author: ^^^Nobukiyo Tanaka^^^
Responsible Curator: ^^^Masahiro Nakajima^^^

Glycoside Hydrolase Family GH162
Clan	GH-x
Mechanism	Inverting
Active site residues	Known
CAZy DB link
http://www.cazy.org/GH162.html

Substrate specificities

In this Glycoside hydrolase family 162, the only β-1,2-glucanase from Talaromyces funiculosus (TfSGL) has been identified, characterized and structurally analyzed to date (5/27/2019) [1]. The enzyme specifically hydrolyzes both cyclic and linear β-1,2-glucans, which comprise a β-linked glucosyl backbone, and preferably releases sophorose (Glc-β-1,2-Glc) from the reducing end of linear β-1,2-glucan [1]. Almost all of the family members are from Eukaryotes [1].

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Kinetics and Mechanism

Hydrolysis of cyclic β-1,2-glucan by TfSGL suggests that the enzyme is an endo-type [1]. The ¹H-NMR analysis of the anomeric configurations of hydrolysates indicates that TfSGL has an inverting mechanism. In addition, the analysis of the change of the degree of optical rotation during the hydrolysis of β-1,2-glucan also supported this mechanism [1]. Structural analysis (see “Three-dimensional structures”) and mutational analysis suggest that D446 is a general base and activates the nucleophilic water via another water [1]. Furthermore, it is predicted that D177 and/or E262 act as a general acid via the 3-hydroxy groups of the Glc moieties (see below) [1]. The result of the action pattern analysis of β-1,2-sophoropentaose derivatives, deoxygenated at their 3-hydroxy groups at the first or second Glc moiety from the reducing end, indicating that E262 act as a general acid via the 3-hydroxy group of the Glc moiety at subsite +2 [1]. The reaction mechanism of TfSGL is quite unique in that both reaction pathways involving a general acid and a general base [1].

Catalytic Residues

The general acid and base of TfSGL are E262 and D446, respectively [1]. Both residues are highly conserved in GH162 enzymes. The general acid of TfSGL is well superimposed with an acidic residue in a GH144 bacterial β-1,2-glucanase from Chitinophaga pinensis (CpSGL), whereas the general base is not superimposed [1, 2]. Although the reaction mechanisms of GH144 are unclear, TfSGL is clearly different from GH144 in the reaction mechanism based on structural comparison [1].

Three-dimensional structures

The apo-structure of the recombinant TfSGL (TfSGLr) was determined at 2.0 Å using the iodide single-wavelength anomalous diffraction phasing method (PDB 6IMU) [1]. The overall structure comprises an (α/α)₆ toroid fold [1]. The complex structure with sophorose (PDB 6IMV) and the Michaelis complex of an inactive TfSGLr-mutant (E262Q) with a β-1,2-glucoheptasaccharide (PDB 6IMW) were also determined by soaking of crystals in sophorose and β-1,2-glucan, respectively [1]. TfSGLr has a cleft crossing the surface of the structure and there is a large active pocket at the center of the cleft [1]. Interestingly, although TfSGL and GH144 enzymes are quite different in their amino acid sequences, their overall structures and the shapes of their active pocket are similar [1]. TfSGLr has slight structural similarity to GH15 and GH8 enzymes.

Family Firsts

First stereochemistry determination: A fungal β-1,2-glucanase from Talaromyces funiculosus by the NMR analysis and the analysis of the change of the degree of optical rotation [1].

First general acid residue identification: A fungal β-1,2-glucanase from Talaromyces funiculosus by the structural analysis, the mutational analysis and the action pattern analysis of β-1,2-sophoropentaose derivatives [1].
First general base residue identification: A fungal β-1,2-glucanase from Talaromyces funiculosus by the structural analysis and the mutational analysis [1].
First 3-D structure: A fungal β-1,2-glucanase from Talaromyces funiculosus using the iodide single-wavelength anomalous diffraction phasing method [1].

References

All Medline abstracts: PubMed

@@ Line 34: / Line 34: @@
 == Kinetics and Mechanism ==
-The analysis of action patterns on cyclic β-1,2-glucan reveal that ''Tf''SGL is an ''endo''-type <cite>Tanaka2019</cite>. The NMR analysis of the anomeric configurations of hydrolysates indicated that ''Tf''SGL has an inverting mechanism. In addition, the analysis of the change of the degree of optical rotation during the hydrolysis of β-1,2-glucan also supported this mechanism <cite>Tanaka2019</cite>.
+Hydrolysis of cyclic β-1,2-glucan by ''Tf''SGL suggests that the enzyme is an ''endo''-type <cite>Tanaka2019</cite>. The <sup>1</sup>H-NMR analysis of the anomeric configurations of hydrolysates indicates that ''Tf''SGL has an inverting mechanism. In addition, the analysis of the change of the degree of optical rotation during the hydrolysis of β-1,2-glucan also supported this mechanism <cite>Tanaka2019</cite>.
-According to the structural analysis (see “Three-dimensional structures”), mutational analysis, D446 (general base) activates the nucleophilic water via another water <cite>Tanaka2019</cite>. Furthermore, it is predicted that D177 and/or E262 act as a general acid via the 3-hydroxy groups of the Glc moieties (see below) <cite>Tanaka2019</cite>. The result of the action pattern analysis of β-1,2-sophoropentaose derivatives, deoxygenated at their 3-hydroxy groups at the first or second Glc moiety from the reducing end, indicating that E262 act as a general acid via the 3-hydroxy group of the Glc moiety at subsite +2 <cite>Tanaka2019</cite>. The reaction mechanism of ''Tf''SGL is quite unique in that both reaction pathways involving a general acid and a general base <cite>Tanaka2019</cite>.
+Structural analysis (see “Three-dimensional structures”) and mutational analysis suggest that D446 is a general base and activates the nucleophilic water via another water <cite>Tanaka2019</cite>. Furthermore, it is predicted that D177 and/or E262 act as a general acid via the 3-hydroxy groups of the Glc moieties (see below) <cite>Tanaka2019</cite>. The result of the action pattern analysis of β-1,2-sophoropentaose derivatives, deoxygenated at their 3-hydroxy groups at the first or second Glc moiety from the reducing end, indicating that E262 act as a general acid via the 3-hydroxy group of the Glc moiety at subsite +2 <cite>Tanaka2019</cite>. The reaction mechanism of ''Tf''SGL is quite unique in that both reaction pathways involving a general acid and a general base <cite>Tanaka2019</cite>.
 == Catalytic Residues ==
-The general acid and base of ''Tf''SGL are E262 and D446, respectively <cite>Tanaka2019</cite>. Both residues are highly conserved in [[GH162]] enzymes. The general acid of ''Tf''SGL is well superimposed with an acidic residue in a [[GH144]] bacterial β-1,2-glucanase from ''Chitinophaga pinensis'' (''Cp''SGL), whereas the general base is not superimposed <cite>Tanaka2019, Abe2017</cite>. Although the reaction mechanisms of GH144 are unclear, ''Tf''SGL is clearly different from [[GH144]] in the reaction mechanism based on structural comparison <cite>Tanaka2019</cite>.
+The general acid and base of ''Tf''SGL are E262 and D446, respectively <cite>Tanaka2019</cite>. Both residues are highly conserved in [[GH162]] enzymes. The general acid of ''Tf''SGL is well superimposed with an acidic residue in a [[GH144]] bacterial β-1,2-glucanase from ''Chitinophaga pinensis'' (''Cp''SGL), whereas the general base is not superimposed <cite>Tanaka2019, Abe2017</cite>. Although the reaction mechanisms of [[GH144]] are unclear, ''Tf''SGL is clearly different from [[GH144]] in the reaction mechanism based on structural comparison <cite>Tanaka2019</cite>.
 == Three-dimensional structures ==
-The apo-structure of the recombinant ''Tf''SGL (''Tf''SGLr) was determined at 2.0 Å using the iodide single-wavelength anomalous diffraction phasing method (PDB 6IMU) <cite>Tanaka2019</cite>. The overall structure comprises an (α/α)<sub>6</sub> toroid fold <cite>Tanaka2019</cite>. The complex structure with sophorose (PDB 6IMV) and the Michaelis complex of an inactive ''Tf''SGLr-mutant (E262Q) with a β-1,2-glucoheptasaccharide (PDB 6IMW) were also determined by soaking of crystals in sophorose and β-1,2-glucan, respectively <cite>Tanaka2019</cite>. ''Tf''SGLr has a cleft crossing the surface of the structure and there is a large active pocket at the center of the cleft <cite>Tanaka2019</cite>. Interestingly, although ''Tf''SGL and GH144 enzymes are quite different in their amino acid sequences, their overall structures and the shapes of their active pocket are similar <cite>Tanaka2019</cite>. ''Tf''SGLr has slight structural similarity to [[GH15]] and [[GH8]] enzymes.
+The apo-structure of the recombinant ''Tf''SGL (''Tf''SGLr) was determined at 2.0 Å using the iodide single-wavelength anomalous diffraction phasing method (PDB 6IMU) <cite>Tanaka2019</cite>. The overall structure comprises an (α/α)<sub>6</sub> toroid fold <cite>Tanaka2019</cite>. The complex structure with sophorose (PDB 6IMV) and the Michaelis complex of an inactive ''Tf''SGLr-mutant (E262Q) with a β-1,2-glucoheptasaccharide (PDB 6IMW) were also determined by soaking of crystals in sophorose and β-1,2-glucan, respectively <cite>Tanaka2019</cite>. ''Tf''SGLr has a cleft crossing the surface of the structure and there is a large active pocket at the center of the cleft <cite>Tanaka2019</cite>. Interestingly, although ''Tf''SGL and [[GH144]] enzymes are quite different in their amino acid sequences, their overall structures and the shapes of their active pocket are similar <cite>Tanaka2019</cite>. ''Tf''SGLr has slight structural similarity to [[GH15]] and [[GH8]] enzymes.
 == Family Firsts ==