Glycoside Hydrolase Family 186

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• Author: Sei Motouchi
• Responsible Curator: Masahiro Nakajima

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Glycoside Hydrolase Family GH186
Clan	None
Mechanism	Inverting
Active site residues	Asp
CAZy DB link
https://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]. Subsequently, GH186 homolog from Xanthomonas campestris pv. campestris (XccOpgD) was found to be an anomer-inverting transglycosylase which specifically generated α-1,6-glucosidic bonds from β-1,2-glucan donors [2]. EcOpgD and XccOpgD are specific toward β-1,2-glucan and the amino acid residues for recognizing β-1,2-glucan at common subsites between EcOpgD and XccOpgD (subsite –7 to +6) are highly conserved in GH186 [1, 2]. However, the reaction types of EcOpgD and XccOpgD are different from each other [1, 2]. EcOpgD is a β-1,2-glucanase that preferentially generates β-1,2-glucooligosaccharides (Sopns, where n indicates a 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]. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan [2]. Almost all family members are found in Pseudomonadota, particularly gamma proteobacteria.

Kinetics and Mechanism

Figure 1. Catalytic center of EcOpgD

Figure 2. Catalytic center of XccOpgD

Optical rotation and NMR analyses indicated that EcOpgD and XccOpgD use anomer-inverting mechanisms [1, 2]. X-ray structural analysis (see below) and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids (Figure 1, 2) [1, 2]. These analyses also suggested that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, act as as general bases to activate the nucleophile via a chain comprising the 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules [1, 2]. In EcOpgD (glucanase), the nucleophile is a water molecule and in XccOpgD (transglycosylase) the nucleophile is the 6-hydroxy group of the Glc moiety at subsite –16 [1, 2].

The difference in nucleophiles among GH186 family is probably due to how the nucleophile and the Grotthuss proton relay are stabilized. While W441, which is important for stabilizing nucleophilic water in EcOpgD, is not conserved in GH186, W76, which is important for stacking with the acceptor Glc moiety in XccOpgD is broadly conserved (but not in the clade of EcOpgD) (Figure 2) [1, 2]. Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, the Grotthuss proton relay is sequestered (stabilized) not by amino acid sequence but by positioning of the acceptor substrate in XccOpgD, with the result that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This explains why XccOpgD is a specific transglycosylase [2].

Catalytic Residues

As described above, the identities of the catalytic residues were indicated by X-ray crystallography and supported by site-directed mutagenesis. The general acid and general base of EcOpgD are D388 and D300, respectively, and the equivalent catalytic residues of XccOpgD are D379 and D291, respectively) [1, 2].

Three-dimensional structures

The ligand-free structure of OpgG from E. coli (EcOpgG) was determined at 2.4 Å (PDB: 1txk) [3]. The ligand-free structure of EcOpgD was determined at 2.95 Å (PDB: 8IOX) [1]. Michaelis complexes of EcOpgD (D388N, co-crystal), EcOpgG (D361N, soaking) and XccOpgD (D379N, soaking) with β-1,2-glucan were determined at 2.06, 1.81, 2.25 Å, respectively (PDB: 8IP1, 8IP2, 8X18) [1, 2].

Figure 3. The overall Michaelis complex structure of EcOpgG (monomer)

There is no structural homolog of GH186 in the 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) (Figure 3). The two domains are connected with one turn of 3₁₀ helix [1, 3]. 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]. In addition, the Loop A of XccOpgD is too short to reach the catalytic center, making the space for recognizing an acceptor β-1,2-glucooligosaccharide moiety [2].

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 [3].

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. Motouchi S, Komba S, Nakai H, and Nakajima M. (2024). Discovery of Anomer-Inverting Transglycosylase: Cyclic Glucohexadecaose-Producing Enzyme from Xanthomonas, a Phytopathogen. J Am Chem Soc. 2024;146(26):17738-17746. DOI:10.1021/jacs.4c02579 |
3. 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 |