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Glycoside Hydrolase Family 186

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Glycoside Hydrolase Family GH186
Clan GH-x
Mechanism inverting
Active site residues Asp
CAZy DB link

Substrate specificities

The defining member of GH186, a β-1,2-glucanase from Escherichia coli (EcOpgD) was identified, characterized and structurally analyzed as reported in 2023[1].EcOpgD is specific toward β-1,2-glucan and the amino acid residues for recognizing β-1,2-glucan are highly conserved in GH186[1]. EcOpgD preferentially generate β-1,2-glucooligosaccharides (Sopns, n is degree of polymerization, DP) with DPs of 6 and 7 from linear β-1,2-glucan[1]. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher[1]. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. Functionally important residues in EcOpgD are not conserved in most of GH186 homologs[1].

Kinetics and Mechanism

Catalytic center of EcOpgD.jpg

Optical rotation analysis indicates that EcOpgD adopt anomer-inverting hydrolytic mechanism[1]. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD directly protonates the scissile glycoside bond as general acid[1]. These analyses also suggest that D300 in EcOpgD activates the nucleophilic water via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general base[1]. Thus, EcOpgD has unique long proton transfer pathway from nucleophilic water to general base.

Catalytic Residues

General acid and base of EcOpgD are D388 and D300, respectively[1].

Three-dimensional structures

The ligand-free structure of OpgG from E. coli (EcOpgG) was determined at 2.5 Å (PDB: 1txk)[2]. The ligand-free structure of EcOpgD was determined at 2.95 Å (PDB: 8IOX)[1]. Michaelis complexes of EcOpgD (D388N, co-crystal) and EcOpgG (D361N, soaking) with β-1,2-glucan were determined at 2.06, 1.81 Å, respectively (PDB: 8IP1, 8IP2)[1].

The overall structure of Michaelis complex of EcOpgG (monomer).jpg

There is no structural homolog of GH186 in whole GH families[1] (January 2024). EcOpgG consists of an N-terminal domain (residues 22–388, β-sandwich) and a C-terminal domain (residues 401–511, Ig-like fold). The two domains are connected with one turn of 310 helix[1, 2]. The loop region (residues 409-425, Loop A below) in the C-terminal domain of the ligand-free structure changes into β-strands in the Michaelis complex structure. In the Michaelis complex, the β-strands reach for the catalytic center of another chain in the dimer to cover the proton transfer pathway from a nucleophile to the general base catalyst[1]. However, the sequence of Loop A is diversified in GH186 family. Indeed, Loop A in EcOpgD sequesters the proton transfer pathway from the solvent, while that of EcOpgG does not completely, which is consistent with the drastically reduced hydrolytic activity of EcOpgG compared with EcOpgD[1].

Family Firsts

First stereochemistry determination
EcOpgD by optical rotation[1].
First general acid residue identification
EcOpgD by X-ray crystallography and site-directed mutagenesis[1].
First general base residue identification
EcOpgD by X-ray crystallography and site-directed mutagenesis[1].
First 3-D structure
EcOpgG by X-ray crystallography[2].


  1. Motouchi S, Kobayashi K, Nakai H, and Nakajima M. (2023). Identification of enzymatic functions of osmo-regulated periplasmic glucan biosynthesis proteins from Escherichia coli reveals a novel glycoside hydrolase family. Commun Biol. 2023;6(1):961. DOI:10.1038/s42003-023-05336-6 | PubMed ID:37735577 [MotouchiEc2023]
  2. Hanoulle X, Rollet E, Clantin B, Landrieu I, Odberg-Ferragut C, Lippens G, Bohin JP, and Villeret V. (2004). Structural analysis of Escherichia coli OpgG, a protein required for the biosynthesis of osmoregulated periplasmic glucans. J Mol Biol. 2004;342(1):195-205. DOI:10.1016/j.jmb.2004.07.004 | PubMed ID:15313617 [Hanoulle2004]

All Medline abstracts: PubMed