Glycoside Hydrolase Family 186

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• Author: Sei Motouchi
• Responsible Curator: Masahiro Nakajima

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Glycoside Hydrolase Family GH186
Clan	GH-x
Mechanism	inverting
Active site residues	known
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 2.4 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]. In EcOpgG, the N-terminal domain (residues 22–388, β-sandwich), which includes about 75% of the protein, is connected to the C-terminal domain (residues 401–511, Ig-like fold) by one turn of 3₁₀ helix[1, 2]. The loop region (residues 409-425, Loop A below) at ligand-free structure on C-terminal domain is changed to β-strand in Michaelis complex structure, and is inserted to the catalytic center of another chain in dimer and interacts with the proton transfer pathway from nucleophile to general base[1]. But 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, which is consistent with the consequence of high and low hydrolytic activities of EcOpgD and EcOpgG[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

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 |
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 |