https://www.cazypedia.org/api.php?action=feedcontributions&feedformat=atom&user=Sei+MotouchiCAZypedia - User contributions [en-ca]2026-04-30T13:19:30ZUser contributionsMediaWiki 1.35.10https://www.cazypedia.org/index.php?title=User:Sei_Motouchi&diff=18253User:Sei Motouchi2024-07-09T06:30:30Z<p>Sei Motouchi: </p>
<hr />
<div>I am a Ph. D candidate at Tokyo university of science. My research interests concern structure and function of new CAZymes, especially β-1,2-glucan-related enzymes responsible for enabling interactions between organisms. I contributed to the biochemical identification and/or structural determination of<br />
<br />
[[GH186]] ''Escherichia coli'' endo β-1,2-glucanase (EcOpgD) '''Family first''' <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of unique reaction mechanism)<br />
<br />
[[GH186]] ''Escherichia coli'' endo β-1,2-glucanase (EcOpgG) <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification and determination of Michaelis complex)<br />
<br />
[[GH186]] ''Xanthomonas campestris'' pv. ''campestris'' α-1,6-cyclized β-1,2-glucohexadecaose synthase (XccOpgD) '''First-identified anomer-inverting transglycosylase''' <cite>Motouchi2024</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of reaction mechanism)<br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Motouchi2023 pmid=37735577 <br />
#Motouchi2024 pmid=38957137 <br />
<br />
</biblio><br />
<br />
<!-- Do not remove this Category tag --><br />
[[Category:Contributors|Motouchi,Sei]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18252Glycoside Hydrolase Family 1862024-07-09T04:05:10Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|None<br />
|-<br />
|'''Mechanism'''<br />
|Inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb|'''Figure 1. Catalytic center of EcOpgD''']][[File:XccOpgD.png|thumb|'''Figure 2. Catalytic center of XccOpgD''']]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis 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''')<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD) ('''Figure 2''')<cite>Motouchi2023 Motouchi2024</cite>. Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, the Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation<cite>Motouchi2024</cite>.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb|'''Figure 3. The overall Michaelis complex structure of EcOpgG (monomer)''']]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18251Glycoside Hydrolase Family 1862024-07-09T03:47:20Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|None<br />
|-<br />
|'''Mechanism'''<br />
|Inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb|'''Figure 1. Catalytic center of EcOpgD''']][[File:XccOpgD.png|thumb|'''Figure 2. Catalytic center of XccOpgD''']]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis 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''')<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD) ('''Figure 2''')<cite>Motouchi2023 Motouchi2024</cite>. Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, the Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation<cite>Motouchi2024</cite>.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb|'''Figure 3. The overall Michaelis complex structure of EcOpgG (monomer)''']]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18217Glycoside Hydrolase Family 1862024-07-08T05:42:14Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|None<br />
|-<br />
|'''Mechanism'''<br />
|Inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb]][[File:XccOpgD.png|thumb]]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD)<cite>Motouchi2023 Motouchi2024</cite>. Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, the Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation<cite>Motouchi2024</cite>.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18216Glycoside Hydrolase Family 1862024-07-08T05:31:35Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|None<br />
|-<br />
|'''Mechanism'''<br />
|Inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
The defining member of GH186, a β-1,2-glucanse from ''Escherichia coli'' (EcOpgD) was identified, characterized and structurally analyzed as reported in 2023<cite>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb]][[File:XccOpgD.png|thumb]]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD)<cite>Motouchi2023 Motouchi2024</cite>. Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, the Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation<cite>Motouchi2024</cite>.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=User:Sei_Motouchi&diff=18215User:Sei Motouchi2024-07-08T03:17:06Z<p>Sei Motouchi: </p>
<hr />
<div>[[Image:Blank_user-200px.png|200px|right]]<br />
<br />
I am a Ph. D candidate at Tokyo university of science. My research interests concern structure and function of new CAZymes, especially β-1,2-glucan-related enzymes responsible for enabling interactions between organisms. I contributed to the biochemical identification and/or structural determination of<br />
<br />
[[GH186]] ''Escherichia coli'' endo β-1,2-glucanase (EcOpgD) '''Family first''' <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of unique reaction mechanism)<br />
<br />
[[GH186]] ''Escherichia coli'' endo β-1,2-glucanase (EcOpgG) <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification and determination of Michaelis complex)<br />
<br />
[[GH186]] ''Xanthomonas campestris'' pv. ''campestris'' α-1,6-cyclized β-1,2-glucohexadecaose synthase (XccOpgD) '''First-identified anomer-inverting transglycosylase''' <cite>Motouchi2024</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of reaction mechanism)<br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Motouchi2023 pmid=37735577 <br />
#Motouchi2024 pmid=38957137 <br />
<br />
</biblio><br />
<br />
<!-- Do not remove this Category tag --><br />
[[Category:Contributors|Motouchi,Sei]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18214Glycoside Hydrolase Family 1862024-07-08T03:03:52Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|None<br />
|-<br />
|'''Mechanism'''<br />
|Inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
The defining member of GH186, a β-1,2-glucanse from ''Escherichia coli'' (EcOpgD) was identified, characterized and structurally analyzed as reported in 2023<cite>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb]][[File:XccOpgD.png|thumb]]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD). Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, the Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18213Glycoside Hydrolase Family 1862024-07-08T02:59:01Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|None<br />
|-<br />
|'''Mechanism'''<br />
|Inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
The defining member of GH186, a β-1,2-glucanse from ''Escherichia coli'' (EcOpgD) was identified, characterized and structurally analyzed as reported in 2023<cite>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb]][[File:XccOpgD.png|thumb]]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD). Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, The Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18212Glycoside Hydrolase Family 1862024-07-08T02:46:59Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
The defining member of GH186, a β-1,2-glucanse from ''Escherichia coli'' (EcOpgD) was identified, characterized and structurally analyzed as reported in 2023<cite>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond<cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. Functionally important residues in EcOpgD and XccOpgD are not conserved in most of GH186 homologs<cite>Motouchi2023 Motouchi2024</cite>. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb]][[File:XccOpgD.png|thumb]]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family is probably due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, while W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD). Therefore, GH186 seems to be fundamentally an anomer-inverting transglycosylase family. In addition, The Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, resulting that a water molecule is not suitable as a nucleophile for efficient Grotthuss proton relay. This is the reason why XccOpgD is specific to transglycosylation.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in the whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>Motouchi2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>Motouchi2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=18210Glycoside Hydrolase Family 1862024-07-05T06:54:28Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
The defining member of GH186, a β-1,2-glucanse from ''Escherichia coli'' (EcOpgD) was identified, characterized and structurally analyzed as reported in 2023<cite>Motouchi2023</cite>. Subsequently GH186 homolog from ''Xanthomonas campestris'' pv. ''campestris'' (XccOpgD) was found to be anomer-inverting transglycoslyase which specifically substitute β-1,2-glucosidic bond of β-1,2-glucan to α-1,6-glucosidic bond at <cite>Motouchi2024</cite>. 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 are highly conserved in GH186<cite>Motouchi2023 Motouchi2024</cite>. However, the reaction types of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. EcOpgD is a β-1,2-glucanase and 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<cite>Motouchi2023</cite>. Final products produced by EcOpgD are Sop6–10, indicating that EcOgpD hydrolyzes Sopns with DPs of 11 and higher<cite>Motouchi2023</cite>. XccOpgD generates only α-1,6-cyclized β-1,2-glucohexadecaose from linear β-1,2-glucan<cite>Motouchi2024</cite>. Almost all family members are found in Pseudomonadota, especially in gamma proteobacteria. Functionally important residues in EcOpgD and XccOpgD are not conserved in most of GH186 homologs<cite>Motouchi2023 Motouchi2024</cite>. <br />
== Kinetics and Mechanism ==<br />
[[File:EcOgpD.png|thumb]][[File:XccOpgD.png|thumb]]Optical rotation and NMR analyses indicate that EcOpgD and XccOpgD adopt anomer-inverting mechanism<cite>Motouchi2023 Motouchi2024</cite>. X-ray structural analysis and mutational analysis suggest that D388 in EcOpgD and the equivalent residue in XccOpgD, D379, directly protonate the scissile glycosidic bond as general acids<cite>Motouchi2023 Motouchi2024</cite>. These analyses also suggest that D300 in EcOpgD and the equivalent residue in XccOpgD, D291, activate the nucleophile via 4-hydroxy group of the Glc moiety at subsite –1 and two water molecules as general bases<cite>Motouchi2023 Motouchi2024</cite>. Interestingly, nucleophiles of EcOpgD and XccOpgD are different from each other<cite>Motouchi2023 Motouchi2024</cite>. The nucleophiles of EcOpgD and XccOpgD are a water molecule and the 6-hydroxy group of Glc moiety at subsite –16, respectively<cite>Motouchi2023 Motouchi2024</cite>. The difference in nucleophile among GH186 family may be due to how a nucleophile and a following Grotthuss proton relay route are stabilized by residues and/or ligands.<br />
<br />
Particularly, While W441 important for stabilizing nucleophilic water in EcOpgD is not conserved in GH186, W76 important for stacking acceptor Glc moiety in XccOpgD is broadly conserved in GH186 (but not in the clade of EcOpgD) in terms of the ability for stacking interaction. Therefore, GH186 seems fundamentally an anomer-inverting transglycosylase family. In addition, The Grotthuss proton relay pathway is sequestered (stabilized) not by amino acid sequence but by acceptor substrate moiety in XccOpgD, meaning that a water molecule is not suitable as nucleophile for efficient Grotthuss proton relay. This is probably the reason why XccOpgD is specific to transglycosylation.<br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively, and the catalytic residues of XccOpgD are also equivalent to that of EcOpgD (D379 and D291, respectively)<cite>Motouchi2023 Motouchi2024</cite>.<br />
== Three-dimensional structures ==<br />
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>Motouchi2023</cite>. 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)<cite>Motouchi2023 Motouchi2024</cite>.<br />
[[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in whole GH families<cite>Motouchi2023</cite> (January 2024).<br />
<br />
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 3<sub>10</sub> helix<cite>Hanoulle2004 Motouchi2023</cite>. 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<cite>Motouchi2023</cite>. 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<cite>Motouchi2023</cite>.In addition, the Loop A of XccOpgD is too short to reach the catalytic center, making the space for recoginizing acceptor β-1,2-glucooligosaccharide moiety<cite>Motouchi2024</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
== References ==<br />
<biblio><br />
#Motouchi2023 pmid=37735577<br />
#Motouchi2024 pmid=38957137<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=File:XccOpgD.png&diff=18209File:XccOpgD.png2024-07-05T06:44:02Z<p>Sei Motouchi: </p>
<hr />
<div>Catalytic center of XccOpgD (inverting transglycosylase)</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=File:EcOgpD.png&diff=18208File:EcOgpD.png2024-07-05T06:43:17Z<p>Sei Motouchi: </p>
<hr />
<div>Catalytic center of EcOpgD</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=File:Catalytic_centers_of_GH186_2.png&diff=18207File:Catalytic centers of GH186 2.png2024-07-05T06:24:17Z<p>Sei Motouchi: </p>
<hr />
<div>Catalytic centers of EcOpgD (β-1,2-glucanase) and XccOpgD (anomer-inverting transglycosylase)</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=File:Catalytic_centers_of_GH186.png&diff=18206File:Catalytic centers of GH186.png2024-07-05T06:12:15Z<p>Sei Motouchi: </p>
<hr />
<div>Catalytic centers of EcOpgD (β-1,2-glucanase) and XccOpgD (inverting transglycosylase)</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17741Glycoside Hydrolase Family 1862024-01-25T09:41:06Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
[[File:Catalytic center of EcOpgD.jpg|thumb]]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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. [[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in whole GH families<cite>MotouchiEc2023</cite> (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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17740Glycoside Hydrolase Family 1862024-01-25T09:30:56Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
[[File:Catalytic center of EcOpgD.jpg|thumb]]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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. [[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in whole GH families<cite>MotouchiEc2023</cite>. 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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17739Glycoside Hydrolase Family 1862024-01-25T09:23:18Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
[[File:Catalytic center of EcOpgD.jpg|thumb]]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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. [[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]There is no structural homolog of GH186 in whole GH families. 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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17738Glycoside Hydrolase Family 1862024-01-25T09:01:22Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
[[File:Catalytic center of EcOpgD.jpg|thumb]]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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. [[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=File:Catalytic_center_of_EcOpgD.jpg&diff=17737File:Catalytic center of EcOpgD.jpg2024-01-25T09:00:58Z<p>Sei Motouchi: </p>
<hr />
<div>Blue dotted lines reprensent the reaction pathway of EcOpgD</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17736Glycoside Hydrolase Family 1862024-01-25T08:47:34Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. [[File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg|thumb]]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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=File:The_overall_structure_of_Michaelis_complex_of_EcOpgG_(monomer).jpg&diff=17735File:The overall structure of Michaelis complex of EcOpgG (monomer).jpg2024-01-25T08:46:48Z<p>Sei Motouchi: </p>
<hr />
<div>N-terminal domain (residues 22-388) and C-terminal domain (residue 401-511) are shown in light green and purple cartoon, respcetively. β-1,2-Glucan is shown as yellow sticks.</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17733Glycoside Hydrolase Family 1862024-01-25T08:11:56Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. 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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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, and is inserted to the catalytic center of another chain in dimer and interacts with the proton transfer pathway from nucleophile to general base<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17732Glycoside Hydrolase Family 1862024-01-25T08:06:26Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
The ligand-free structure of OpgG from ''E. coli'' (EcOpgG) was determined at 2.5 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>. 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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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, and is inserted to the catalytic center of another chain in dimer and interacts with the proton transfer pathway from nucleophile to general base<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17729Glycoside Hydrolase Family 1862024-01-25T07:45:01Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
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>.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 3<sub>10</sub> helix<cite>Hanoulle2004 MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>. 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<cite>MotouchiEc2023</cite>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_186&diff=17725Glycoside Hydrolase Family 1862024-01-24T08:47:57Z<p>Sei Motouchi: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: [[User:Sei Motouchi|Sei Motouchi]]<br />
* [[Responsible Curator]]: [[User:Masahiro Nakajima|Masahiro Nakajima]]<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH186'''<br />
|-<br />
|'''Clan''' <br />
|GH-x<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH186.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
<br />
== Substrate specificities ==<br />
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>. <br />
== Kinetics and Mechanism ==<br />
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. <br />
== Catalytic Residues ==<br />
General acid and base of EcOpgD are D388 and D300, respectively<cite>MotouchiEc2023</cite>.<br />
== Three-dimensional structures ==<br />
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>.<br />
== Family Firsts ==<br />
;First stereochemistry determination: EcOpgD by optical rotation<cite>MotouchiEc2023</cite>.<br />
;First general acid residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First general base residue identification: EcOpgD by X-ray crystallography and site-directed mutagenesis<cite>MotouchiEc2023</cite>.<br />
;First 3-D structure: EcOpgG by X-ray crystallography<cite>Hanoulle2004</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#MotouchiEc2023 pmid=37735577<br />
#Hanoulle2004 pmid=15313617<br />
<br />
</biblio><br />
<br />
<!-- Do not delete this Category tag --><br />
[[Category:Glycoside Hydrolase Families|GH186]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=User:Sei_Motouchi&diff=17559User:Sei Motouchi2023-10-20T10:45:06Z<p>Sei Motouchi: </p>
<hr />
<div>[[Image:Blank_user-200px.png|200px|right]]<br />
<br />
I am a Ph. D candidate at Tokyo university of science. My research interests concern structure and function of new CAZymes, especially β-1,2-glucan-related enzymes responsible for enabling interactions between organisms. I contributed to the biochemical identification and/or structural determination of<br />
<br />
[[GH186]] ''Escherichia coli'' endo β-1,2-glucanase (EcOpgD) '''Family first''' <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of unique reaction mechanism)<br />
<br />
[[GH186]] ''Escherichia coli'' endo β-1,2-glucanase (EcOpgG) <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification and determination of Michaelis complex)<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Motouchi2023 pmid=37735577 <br />
<br />
</biblio><br />
<br />
<!-- Do not remove this Category tag --><br />
[[Category:Contributors|Motouchi,Sei]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=User:Sei_Motouchi&diff=17558User:Sei Motouchi2023-10-20T10:37:03Z<p>Sei Motouchi: </p>
<hr />
<div>[[Image:Blank_user-200px.png|200px|right]]<br />
<br />
I am a Ph. D candidate at Tokyo university of science. My research interests concern structure and function of new CAZymes, especially β-1,2-glucan-related enzymes responsible for enabling interactions between organisms. I contributed to the biochemical identification and/or structural determination of<br />
<br />
[[GH186]] Escherichia coli endo β-1,2-glucanase (EcOpgD) '''Family first''' <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of unique reaction mechanism)<br />
<br />
[[GH186]] Escherichia coli endo β-1,2-glucanase (EcOpgG) <cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification and determination of Michaelis complex)<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Motouchi2023 pmid=37735577 <br />
<br />
</biblio><br />
<br />
<!-- Do not remove this Category tag --><br />
[[Category:Contributors|Motouchi,Sei]]</div>Sei Motouchihttps://www.cazypedia.org/index.php?title=User:Sei_Motouchi&diff=17557User:Sei Motouchi2023-10-20T10:11:28Z<p>Sei Motouchi: </p>
<hr />
<div>[[Image:Blank_user-200px.png|200px|right]]<br />
<br />
I am a Ph. D candidate at Tokyo university of science. My research interests concern structure and function of new CAZymes, especially β-1,2-glucan-related enzymes responsible for enabling interactions between organisms. I contributed to the biochemical identification and/or structural determination of<br />
<br />
[[GH186]] Escherichia coli endo β-1,2-glucanase (EcOpgD) '''Family first'''<cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification, determination of Michaelis complex and Suggestion of unique reaction mechanism)<br />
<br />
[[GH186]] Escherichia coli endo β-1,2-glucanase (EcOpgG)<cite>Motouchi2023</cite>.<br />
<br />
(biochemical identification and determination of Michaelis complex)<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<biblio><br />
Motouchi2023 Motouchi, S., Kobayashi, K., Nakai, H., Nakajima, M. Identification of enzymatic functions of osmo-regulated periplasmic glucan biosynthesis proteins from Escherichia coli reveals a novel glycoside hydrolase family. Commun Biol 2023 Sep 21;6(1):961. https://doi.org/10.1038/s42003-023-05336-6 |PubMed ID:37735577 <br />
<br />
</biblio><br />
<br />
<!-- Do not remove this Category tag --><br />
[[Category:Contributors|Motouchi,Sei]]</div>Sei Motouchi