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Difference between revisions of "Glycoside Hydrolase Family 186"
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== Substrate specificities == | == 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<cite>MotouchiEc2023</cite>.EcOpgD is specific toward β-1,2-glucan and the amino acid residues for recognizing β-1,2-glucan are highly conserved in GH186<cite>MotouchiEc2023</cite>. EcOpgD preferentially generate β-1,2-glucooligosaccharides (Sop<sub>n</sub>s, n is degree of polymerization, DP) with DPs of 6 and 7 from linear β-1,2-glucan<cite>MotouchiEc2023</cite>. Final products produced by EcOpgD are Sop<sub>6–10</sub>, indicating that EcOgpD hydrolyzes Sop<sub>n</sub>s with DPs of 11 and higher<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. | |
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== Kinetics and Mechanism == | == Kinetics and Mechanism == | ||
| − | + | Optical rotation analysis indicates that EcOpgD adopt anomer-inverting hydrolytic mechanism<cite>MotouchiEc2023</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD directly protonates the scissile glycoside bond as general acid<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. Thus, EcOpgD has unique long proton transfer pathway from nucleophilic water to general base. | |
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== Catalytic Residues == | == Catalytic Residues == | ||
| − | + | General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>. | |
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== Three-dimensional structures == | == Three-dimensional structures == | ||
| − | + | The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.4 Å (PDB: 1txk)<cite>Hanoulle2004</cite>. The ligand-free structure of EcOpgD was determined at 2.95 Å (PDB: 8IOX)<cite>MotouchiEc2023</cite>. 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)<cite>MotouchiEc2023</cite>. | |
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== Family Firsts == | == Family Firsts == | ||
| − | ;First stereochemistry determination: | + | ;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>. |
| − | ;First | + | ;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>. |
| − | ;First general | + | ;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>. |
| − | ;First 3-D structure: | + | ;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>. |
== References == | == References == | ||
<biblio> | <biblio> | ||
| − | # | + | #MotouchiEc2023 pmid=37735577 |
| − | # | + | #Hanoulle2004 pmid=15313617 |
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</biblio> | </biblio> | ||
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[[Category:Glycoside Hydrolase Families|GH186]] | [[Category:Glycoside Hydrolase Families|GH186]] | ||
Revision as of 00:47, 24 January 2024
This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.
| Glycoside Hydrolase Family GH186 | |
| Clan | GH-x |
| Mechanism | inverting |
| Active site residues | known |
| 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].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
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].
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
- 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 |
- 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 |