Glycoside Hydrolase Family 162

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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

Figure 1. The phylogenetic tree of GH162 homologs.

In Glycoside hydrolase family 162, a β-1,2-glucanase only from Talaromyces funiculosus (TfSGL) has been identified, characterized and structurally analyzed as of June 4th, 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]

Kinetics and Mechanism

Figure 2. Structurally possible pathways for a general acid (A) and the reaction mechanism (B). (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 [].

Hydrolysis of cyclic β-1,2-glucan by TfSGL suggests that the enzyme is endo-type [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”) 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 the 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 are unclear as of June 4th, 2019, TfSGL is clearly different from GH144 in the reaction mechanism based on structural comparison [1].

Three-dimensional structures

Figure 3. Overall structure of TfSGL (PDB 6IMU).

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 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].

References

All Medline abstracts: PubMed