Glycoside Hydrolase Family 144

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• Author: Koichi Abe
• Responsible Curator: Masahiro Nakajima

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Glycoside Hydrolase Family GH144
Clan	GH-S
Mechanism	Inverting
Active site residues	Not known
CAZy DB link
https://www.cazy.org/GH144.html

Substrate specificities

Glycoside hydrolase family 144 (GH144) contains β-1,2-glucan-hydrolyzing enzymes. The first characterized enzymes of this family are an endo-β-1,2-glucanase (EC 3.2.1.71) from Chitinophaga pinensis (CpSGL) and a sophorohydrolase (nonreducing end) (EC 3.2.1.-) from Parabacteroides distasonis (BDI_3064) [1, 2]. CpSGL hydrolyzes β-1,2-glucan and mainly releases β-1,2-glucooligosaccharides with degrees of polymerization (DPs) of 3–5, whereas BDI_3064 efficiently hydrolyzes shorter β-1,2-glucooligosaccarides with DPs of more than 3 to produce sophorose (Glc-β-1,2-Glc) from the nonreducing end of β-1,2-glucooligosaccarides. These enzyme are highly specific for β-1,2-glucan or its shorter oligosaccharides.

Kinetics and Mechanism

CpSGL hydrolyzes cyclic β-1,2-glucan, showing that this enzyme is endo-hydrolytic, whereas BDI_3064 does not, indicating that this enzyme is exo-hydrolytic [1, 2]. Monitoring the stereochemical course of the hydrolysis of β-1,2-glucan by ¹H-NMR showed that CpSGL uses an inverting mechanism to hydrolyze β-1,2-glucan [1]. Monitoring the change of the degree of optical rotation during hydrolysis of β-1,2-glucan by CpSGL also supported this mechanism [1].

Catalytic Residues

Mutational analysis of CpSGL indicated that Asp139, Glu142, and Glu211 play important roles in catalysis [1]. However, structural analysis of CpSGL showed that none of these residues are in positions that can directly transfer hydrogen to the O2 atoms of the glucose moieties in sophorotriose, nor are they proximal to the space between the bound glucose and sophorotriose. Comparison of topological positions of the conserved acidic residues in CpSGL with the catalytic residues in inverting GHs with a similar fold (GH8, GH15, and GH162) did not provide clues to the assignment of the catalytic residues. These observation may imply that GH144 enzymes have a non-canonical reaction mechanism.

Three-dimensional structures

The 3-D structures of CpSGL (PDB IDs 5GZH and 5GZK) and BDI_3064 (PDB ID 5Z06) were determined by X-ray crystallography and revealed the (α/α)₆-fold of this family [1]. BDI_3064 possesses additional N-terminal domains 1 and 2, important for the substrate specificity of this enzyme, as described below. The overall structure of CpSGL is similar to that of GH162 endo-β-1,2-glucanase (TfSGL) despite their low sequence similarity [3].

The crystal structure of CpSGL in complex with glucose and sophorotriose provided the structural basis for substrate recognition of this enzyme. CpSGL possesses the large cleft typical of endo-acting enzymes. HPLC and ESI-MS analysis suggested that the bound glucose and sophorotriose occupies −3 subsite and +1 to +3 subsites, respectively. The ligand-free crystal structure and docking analysis of BDI_3064 showed that Arg93 in the N-terminal domain 1 overlaps −3 subsite and completely blocks the non-reducing end of the docked β-1,2-glucooligosaccharides [2]. This structural feature would make BDI_3064 an exo-acting enzyme.

The unliganded crystal structures of GH144 enzymes from Bacteroides species (PDB IDs 3EU8, 4GL3, and 4QT9) were deposited in the PDB database before the deposition of that of CpSGL. However, these Bacteroides enzymes have not been biochemically characterized. Later, GH144 is classified into clan GH-S with GH162 based on structural similarity between them [4].

Family Firsts

First stereochemisty determination

Monitoring hydrolysis of β-1,2-glucan by ¹H-NMR spectroscopy and polarimetric analysis showed that CpSGL hydrolyzes β-1,2-glucan with inversion of stereochemistry.

First general acid residue identification

Not known.

First general base residue identification

Not known.

First 3-D structure

The first deposited protein in the PDB database is BF9343_0330 protein from Bacteroides fragilis NCTC 9343 (PDB ID 3EU8) followed by the deposition of BACUNI_03963 and BACCAC_03554 proteins from Bacteroides species (PDB IDs 4GL3 and 4QT9, respectively). These structures were determined by Joint Center for Structural Genomics in ligand-free form. However, there are no journal publications describing these proteins. The first published structures are those of CpSGL (PDB IDs 5GZH and 5GZK), one of which (5GZH) captures sophorotriose and glucose.

References

1. Abe K, Nakajima M, Yamashita T, Matsunaga H, Kamisuki S, Nihira T, Takahashi Y, Sugimoto N, Miyanaga A, Nakai H, Arakawa T, Fushinobu S, and Taguchi H. (2017). Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family. J Biol Chem. 2017;292(18):7487-7506. DOI:10.1074/jbc.M116.762724 |
2. Shimizu H, Nakajima M, Miyanaga A, Takahashi Y, Tanaka N, Kobayashi K, Sugimoto N, Nakai H, and Taguchi H. (2018). Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β-1,2-Glucooligosaccharides from Parabacteroides distasonis. Biochemistry. 2018;57(26):3849-3860. DOI:10.1021/acs.biochem.8b00385 |
3. 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. 2019;294(19):7942-7965. DOI:10.1074/jbc.RA118.007087 |
4. Tanaka N, Saito R, Kobayashi K, Nakai H, Kamo S, Kuramochi K, Taguchi H, Nakajima M, and Masaike T. (2024). Functional and structural analysis of a cyclization domain in a cyclic β-1,2-glucan synthase. Appl Microbiol Biotechnol. 2024;108(1):187. DOI:10.1007/s00253-024-13013-9 |