Glycoside Hydrolase Family 186

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Glycoside Hydrolase Family GH186
Clan	GH-x
Mechanism	inverting
Active site residues	Asp
CAZy DB link
http://www.cazy.org/GH186.html

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 (Sop_ns, 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 Sop_6–10, indicating that EcOgpD hydrolyzes Sop_ns 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

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].

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].

References

All Medline abstracts: PubMed