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Figure 1. The phylogenetic tree of GH162 homologs.
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 [1]. 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 [1]. Almost all of the family members are from Eukaryotes [1]
Kinetics and Mechanism
Figure 2. Active site and reaction mechanism.(A) The complex of the E262Q mutant with β-1,2-glucoheptaose. The numbers beside the substrate represent the positions of subsites. The red and blue dotted lines represent the hydrogen bonds between the ligands and D177 or E262, respectively. The β-1,2-glucotriose moiety in the observed substrate is represented by a yellow stick. Candidate residues for a general acid are represented by brown sticks. The 262th glutamine residue is represented as a glutamic acid. (B) E262 (general acid) indirectly protonates the glycosidic bond oxygen atom via the 3-hydroxy group of the Glc moiety at subsite +2 and D446 (general base) activates the nucleophilic water via another water [1].
Hydrolysis of cyclic β-1,2-glucan by TfSGL suggests that the enzyme is endo-acting [1]. 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 [1].
Structural analysis (see “Three-dimensional structures” below) and mutational analysis suggest that D446 activates the nucleophilic water via another water as a general acid [1]. 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) [1]. 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 [1]. The reaction mechanism of TfSGL is quite unique in that both reaction pathways involving a general acid and a general base are non-canonical [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 enzymes are currently unclear (June 2019), structural comparison of TfSGL and GH144 suggests differences in reaction mechanisms [1].
A structural comparison revealed that the position of the general acid residue in GH162 and the candidate catalytic residue in GH144 are well superimposed structurally [3]. In contrast, the positions of the other catalytic residues (or candidate catalytic residues) in GH162 and GH144 are completely different [3]. Furthermore, compared to clans GH-G, L, M, O, P and Q, which have the same overall structure (= (α/α)6 fold) as GH162 and GH144, none of the positions of the catalytic residues in GH162 and GH144 are conserved [3]. A new clan GH-S was created for GH162 and GH144 based on these results [3]. Later, GH192, GH193, and GH194 joined clan GH-S [4].
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 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 [1]. TfSGLr has a cleft crossing the surface of the structure and there is a large active-site 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 positions of the substrates in their catalytic pockets 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].
