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Glycoside Hydrolase Family 162
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|Glycoside Hydrolase Family GH162|
|Active site residues||Known|
|CAZy DB link|
The defining member of glycoside hydrolase family 162, a β-1,2-glucanase from Talaromyces funiculosus (TfSGL), was identified, characterized, and structurally analyzed as reported in 2019 . This 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 . Almost all of the family members are from Eukaryotes 
Kinetics and Mechanism
Hydrolysis of cyclic β-1,2-glucan by TfSGL suggests that the enzyme is endo-acting . The 1H-NMR analysis of the anomeric configurations of hydrolysates indicates that TfSGL has an inverting mechanism. Analysis of the change of the degree of optical rotation during hydrolysis of β-1,2-glucan also supported this mechanism .
Structural analysis (see “Three-dimensional structures” below) and mutational analysis suggest that D446 activates the nucleophilic water via another water as a general acid . These analyses also suggest that D177 and/or E262 act as a general acid via the 3-hydroxy groups of the Glc moieties (see below) . According to action-pattern analysis using β-1,2-glucopentaose derivatives deoxygenated at their 3-hydroxy groups in the first or second Glc moiety from the reducing end, E262 was clearly determined to be a general acid. The 3-hydroxy group of the Glc moiety at subsite +2 mediates protonation of glycosidic bond oxygen atom . The reaction mechanism of TfSGL is quite unique in that both reaction pathways involving a general acid and a general base are non-canonical .
The general acid and base of TfSGL are E262 and D446, respectively . 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 enzymes are currently unclear (June 2019), structural comparison of TfSGL and GH144 suggests differences in reaction mechanisms .
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) . The overall structure comprises an (α/α)6 toroid fold . The complex structures with sophorose (PDB 6IMV) and the Michaelis complex of an inactive TfSGLr-mutant (E262Q) with a β-1,2-glucoheptaose (PDB 6IMW) were also determined by soaking of crystals in sophorose and β-1,2-glucan, respectively . TfSGLr has a cleft crossing the surface of the structure and there is a large active-site pocket at the center of the cleft . Interestingly, although TfSGL and GH144 enzymes are quite different in their amino acid sequences, their overall structures and the positions of the substrates in their catalytic pockets are similar . TfSGLr has slight structural similarity to GH15 and GH8 enzymes.
- 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 .
- 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 .
- First general base residue identification
- A fungal β-1,2-glucanase from Talaromyces funiculosus by the structural analysis and the mutational analysis .
- First 3-D structure
- A fungal β-1,2-glucanase from Talaromyces funiculosus using the iodide single-wavelength anomalous diffraction phasing method .
- Tanaka N, Nakajima M, Narukawa-Nara M, Matsunaga H, Kamisuki S, Aramasa H, Takahashi Y, Sugimoto N, Abe K, Terada T, Miyanaga A, Yamashita T, Sugawara F, Kamakura T, Komba S, Nakai H, and Taguchi H. (2019) Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family. J Biol Chem. 294, 7942-7965. DOI:10.1074/jbc.RA118.007087 |
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