CAZypedia - User contributions [en-ca]

Glycoside Hydrolase Family 128

2020-08-13T05:33:51Z

Camila Santos:

{{UnderConstruction}}
* [[Author]]: ^^^Yuichi Sakamoto^^^ and ^^^Camilla Santos^^^
* [[Responsible Curator]]: ^^^Mario Murakami^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH128'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH128.html
|}
</div>


== Substrate specificities ==
The first GH128 enzyme, GLU1, was cloned from ''Lentinula edodes'' fruiting bodies (shiitake mushroom) <cite>Sakamoto2011</cite>. GLU1 cleaves β-1,3 linkages in various β-glucans such as lentinan from itfself, laminarin from ''Laminaria digitata'', pachyman from ''Poria cocos'', and curdlan from ''Alcaligenes faecalis'' but does not degrade β-1,3-linkages within β-1,3-1,4-glucans such as barley glucan, indicating the enzyme is categorized into EC [{{EClink}}3.2.1.39 3.2.1.39] <cite>Sakamoto2011</cite>. A further work with several GH128 members corroborated that this family is specific for β-1,3-glucans <cite>Santos2020</cite>. In addition, it was showed that bacterial members are endo-β-1,3-glucanases that degrade these carbohydrates at higher rates, such as those from ''Amycolatopsis mediterranei'' (subgroup I) and ''Pseudomonas viridiflava'' (subgroup II) <cite>Santos2020</cite>. Fungal enzymes are more diverse in terms of activity: endo-β-1,3-glucanases, represented by the GLU1 from ''L. edodes'' (subgroup IV) <cite>Sakamoto2011 Santos2020</cite>; exo-β-1,3-glucanases that release trisaccharides (''Aureobsidium namibiae'') (subgroup VI) <cite>Santos2020</cite> and monosaccharides (''Cryptococcus neoformans'') (subgroup V) <cite>Santos2020</cite> from the reducing ends; and exo-β-1,3-glucanases that release trisaccharides from the non-reducing ends of triple-helical β-1,3-glucans, represented by the enzyme from ''Blastomyces gilchristii'' <cite>Santos2020</cite> (subgroup III). Some fungal members from this family are devoid of catalytic activity but conserve the capacity to bind short β-1,3-glucooligosaccharides (subgroup VII) such as those from ''Trichoderma gamsii'' <cite>Santos2020</cite> and ''C. neoformans'' <cite>Santos2020</cite>.
== Kinetics and Mechanism ==
As predicted by the first study of a GH128 enzyme <cite>Sakamoto2011</cite>, this family is part of the Clan GH-A and its members are retaining enzymes, which operate by a classical Koshland retention mechanism as confirmed through <sup>1</sup>H-nuclear magnetic resonance spectroscopy with the retention of the anomeric configuration of enzymatic products <cite>Santos2020</cite>.

== Catalytic Residues ==
From the sequence alignment of GH128 members, two glutamic acids, E103 and E195 in ''L. edodes'' GLU1, were predicted to be the catalytic residues <cite>Sakamoto2011</cite>. They were further confirmed to be the acid/base and the nucleophile, respectively, by site-directed mutagenesis of the bacterial GH128 member from ''A. mediterranei'' <cite>Santos2020</cite>. These residues are located at the C-terminal ends of the strands β7 and β4 <cite>Santos2020</cite>, as observed for other clan GH-A families.

== Three-dimensional structures ==
A three-dimensional homology model of ''L. edodes'' GLU1 indicated similarity with several (β/α)<sub>8</sub>-barrel (TIM-barrel) structures, including a [[GH39]] β-xylosidase and a [[GH5]] β-mannanase <cite>Sakamoto2011</cite>. The fold resembling an (β/α)<sub>8</sub>-barrel was further confirmed with the crystal structure determination of 9 members of the family <cite>Santos2020</cite>. However, in all structures, the helix α2 and the strand β3 are strictly absent <cite>Santos2020</cite>. Moreover, some enzymes such as the endo-β-1,3-glucanase from ‘‘L. edodes’’ (GLU1) and the exo-β-1,3-glucanase from ''C. neoformans'', also lack the helices α1 and α3, respectively <cite>Santos2020</cite>.

Two distinct modes of substrate binding were observed in the GH128 family <cite>Santos2020</cite>. The most widespread mode, named as hydrophobic knuckle, involves a tryptophan residue that interacts with four glucoside moieties from –5 to –2 and is fully complementary to the typically curved conformation of β-1,3-glucan chains. The other mode, only observed in fungal members belonging to subgroups IV and VI, requires substrate conformational changes to allow the binding to the catalytic interface. In these fungal subgroups, the hydrophobic knuckle is absent and two aromatic residues, positioned at the -5 and -4 subsites, create a linearized cleft, which requires a 180° torsion in the glycosidic bond between the glycosyl moieties –2 and –3 in the β-1,3-glucan chain for binding. This mode of substrate recognition is called as “flattening” mechanism due to the unusual conformational, but also stereochemically favorable, adopted by the substrate. It is notable that such mode of substrate binding was not yet observed in other CAZy families active on β-1,3-glucans.

== Clustering of GH128 ==
[[Image:Santos_GH128_final.png|thumb|right|250px|Figure 1. Clustering of the GH128 family into seven subgroups. Adapted from <cite>Santos2020</cite>.]]

The glycoside hydrolase family 128 was created based on the study of Yuichi Sakamoto and colleagues <cite>Sakamoto2011</cite>. Years later, the group headed by Mario Murakami carried out a task force to explore the functional and structural diversity of this family <cite>Santos2020</cite>. For this purpose, they employed phylogenetic and SSN analyses to segregate the family into putative isofunctional subgroups. The SSN analysis resulted in two well discretized clusters (subgroups VI and VII) and a third cluster that was further subdivided into five subgroups (I to V) based on SSN alignment scores and evolutionary closeness (Fig. 1). At least one member of each subgroup was biochemically and structurally characterized: AmGH128_I, PvGH128_II, ScGH128_II, BgGH128_III, LeGH128_IV, CnGH128_V, AnGH128_VI, TgGH128_VII and CnGH128_VII. Subgroups I and II were found to be predominantly present in bacteria, and the subgroups III to VII are mostly found in fungi. Bacterial enzymes are faster, present the hydrophobic knuckle and attack the β-1,3-glucan in an endo mode of action, which is compatible with their biological function: nutrition and competition. Fungal β-1,3-glucanases are known to act on remodeling of their own cell walls. Therefore, these enzymes are slower, more diverse in terms of substrate recognition modes (flattening mechanism – subgroups IV and VI; hydrophobic knuckle – subgroups III, V and VII) and mode of action (exo-enzymes – subgroups III, V and VI; endo-enzymes – subgroup IV; oligosaccharide binding proteins – subgroup VII). This was the first time that a glycoside hydrolase family was rationally studied based on SSN analysis. A recent study led by Prof. Harry Brumer applied a similar strategy to classify the polyspecific GH16 family into isofunctional subgroups using the available functional and structural data in the literature <cite>Viborg2019</cite>, highlighting this approach as a promising strategy to systematically assess the functional and structural diversity of CAZyme families. It is noteworthy to point out that Brumer´s group made available an intuitive and robust program to perform SSN analyses, named as SSNpipe that is freely available from GitHub (https://github.com/ahvdk/SSNpipe).

== Family Firsts ==
;First stereochemistry determination: predicted to be retaining by membership in Clan GH-A <cite>Sakamoto2011</cite> and further validated by <sup>1</sup>H-NMR of products of the ''A. mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.

;First catalytic nucleophile identification: predicted by sequence alignment <cite>Sakamoto2011</cite> and confirmed by site-directed mutagenesis of ''A. mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.

;First general acid/base residue identification: predicted by sequence alignment <cite>Sakamoto2011</cite> and confirmed by site-directed mutagenesis of ''A. mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.

;First 3-D structure: predicted by modelling of ''L. edodes'' GLU1 <cite>Sakamoto2011</cite> and experimentally determined for several GH128 members including endo-β-1,3-glucanases from ''A. mediterranei'' (AmGH128_I), ''P. viridiflava'' (PvGH128_II), ''Sorangium cellulosum'' (ScGH128_II) and ''L. edodes'' (LeGH128_IV); exo-β-1,3-glucanases from ''B. gilchristii'' (BgGH128_III), ''C. neoformans'' (CnGH128_V) and ''A. namibiae'' (AnGH128_VI); and β-1,3-glucooligosaccharide binding proteins from ''T. gamsii'' (TgGH128_VII) and ''C. neoformans'' (CnGH128_VII) <cite>Santos2020</cite>.

== References ==
<biblio>

#Sakamoto2011 pmid=21965406
#Santos2020 pmid=32451508
#Viborg2019 pmid=31501245

</biblio>

[[Category:Glycoside Hydrolase Families|GH128]]

Glycoside Hydrolase Family 128

2020-07-29T17:46:03Z

Camila Santos:

{{UnderConstruction}}
* [[Author]]: ^^^Yuichi Sakamoto^^^ and ^^^Camilla Santos^^^
* [[Responsible Curator]]: ^^^Mario Murakami^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH128'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH128.html
|}
</div>


== Substrate specificities ==
Glycoside hydrolase Family 128 comprises prokaryotic and eukaryotic enzymes that are active on β-1,3-glucans. Endo-β-1,3-glucanases that degrade the carbohydrate at higher rates are found in bacterial subgroups (I and II) such as those from ''Amycolatopsis mediterranei'' <cite>Santos2020</cite> and ''Pseudomonas viridiflava'' <cite>Santos2020</cite>. Fungal enzymes, which are likely involved in cell wall remodeling processes, are more diverse in terms of activity: endo-β-1,3-glucanases, represented by the enzyme from ''Lentinula edodes'' (subgroup IV) <cite>Santos2020 Sakamoto2011</cite>; exo-β-1,3-glucanases that release trisaccharides (''Aureobsidium namibiae'', subgroup VI) <cite>Santos2020</cite> and monosaccharides (''Cryptococcus neoformans'', subgroup V) <cite>Santos2020</cite> from the reducing ends; and exo-β-1,3-glucanases that release trisaccharides from the non-reducing ends of triple-helical β-1,3-glucans, represented by the enzyme from ''Blastomyces gilchristii'' (subgroup III) <cite>Santos2020</cite>. Some fungal members from this family are devoid of catalytic activity but conserve the capacity to bind short β-1,3-glucooligosaccharides (subgroup VII) such as those from ''Trichoderma gamsii'' <cite>Santos2020</cite> and a second GH128 member from ''Cryptococcus neoformans'' <cite>Santos2020</cite>.

== Kinetics and Mechanism ==
Family 128 enzymes are retaining enzymes, which operate by a classical Koshland retention mechanism as confirmed through <sup>1</sup>H-nuclear magnetic resonance spectroscopy with the retention of the anomeric configuration of enzymatic products <cite>Santos2020</cite>.

== Catalytic Residues ==
As a clan GH-A family, the two acidic catalytic residues are located at the C-terminal ends of the strands β7 and β4. Both nucleophile and acid/base are glutamates and their function were validated by site-directed mutagenesis <cite>Santos2020</cite>

== Three-dimensional structures ==
The GH128 members exhibit a fold resembling an (α/β)<sub>8</sub>-barrel in which the helix α2 and the strand β3 are strictly absent <cite>Santos2020</cite>. Some enzymes such as the endo-β-1,3-glucanase from ''Lentinula edodes'' and the exo-β-1,3-glucanase from ''Cryptococcus neoformans'', also lack the helices α1 and α3, respectively <cite>Santos2020</cite>.

Two distinct modes of substrate binding were observed in the GH128 family <cite>Santos2020</cite>. The most widespread mode, named as hydrophobic knuckle, involves a tryptophan residue that interacts with four glucoside moieties from -5 to -2 and is fully complementary to the typically curved conformation of β-1,3-glucan chains. The other mode, only observed in fungal members belonging to subgroups IV and VI, requires substrate conformational changes to allow the binding to the catalytic interface. In these fungal subgroups, the hydrophobic knuckle is absent and two aromatic residues, positioned at the -5 and -4 subsites, create a linearized cleft, which requires a 180° torsion in the glycosidic bond between the glycosyl moieties -2 and -3 in the β-1,3-glucan chain for binding. This mode of substrate recognition is called as “flattening” mechanism due to the unusual conformational, but also stereochemically favorable, adopted by the substrate. It is notable that such mode of substrate binding was not yet observed in other CAZy families active on β-1,3-glucans.
== Clustering of GH128 ==

[[Image:Santos_GH128_final.png|thumb|right|250px|Figure 1. Clustering of the GH128 family into seven subgroups. Adapted from <cite>Santos2020</cite>.]]

After creation of the GH128 family by Y. Sakamoto and colleagues <cite>Sakamoto2011</cite>, the group headed by M. Murakami carried out a task force to explore the functional and structural diversity of this family <cite>Santos2020</cite>. For this purpose, they employed phylogenetic and SSN analyses to segregate the family into putative isofunctional subgroups. The SSN analysis resulted in two well discretized clusters (subgroups VI and VII) and a third cluster that was further subdivided into five subgroups (I to V) based on SSN alignment scores and evolutionary closeness (Fig. 1). Them, they characterized, biochemically and structurally, one or more members of each subgroup: AmGH128_I, PvGH128_II, ScGH128_II, BgGH128_III, LeGH128_IV, CnGH128_V, AnGH128_VI, TgGH128_VII and CnGH128_VII. Subgroups I and II were found to be predominantly present in bacteria, and the subgroups III to VII are mostly found in fungi. Bacterial enzymes are faster, present the hydrophobic knuckle and attack the β-1,3-glucan in an endo mode of action, which is compatible with their biological function: nutrition and competition. Fungal β-1,3-glucanases are known to act on remodeling of their own cell walls. Therefore, these enzymes are slower, more diverse in terms of substrate recognition modes (flattening mechanism - subgroups IV and VI; hydrophobic knuckle - subgroups III, V and VII) and mode of action (exo-enzymes - subgroups III, V and VI; endo-enzymes - subgroup IV; oligosaccharide binding protein - subgroup VII). It was the first time that a glycoside hydrolase family was rationally studied based on SSN analysis. It is noteworthy to mention that a recent study led by Prof. H. Brumer applied a similar strategy to classify the polyspecific GH16 family into isofunctional subgroups using the available functional and structural data in the literature <cite>Viborg2019</cite>. In addition, Brumer´s group made available an intuitive and robust program to perform SSN analyses, named as SSNpipe that is freely available from GitHub.
== Family Firsts ==
;First stereochemistry determination: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) by <sup>1</sup>H-NMR <cite>Santos2020</cite>.
;First catalytic nucleophile identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First general acid/base residue identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First 3-D structure: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.
== References ==
<biblio>
#Santos2020 pmid=32451508
#Sakamoto2011 pmid=21965406
#Viborg2019 pmid=31501245

</biblio>

[[Category:Glycoside Hydrolase Families|GH128]]

File:Santos GH128 final.png

2020-07-29T17:45:19Z

Camila Santos:

File:Santos GH128 fig2.png

2020-07-29T16:34:16Z

Camila Santos: Camila Santos uploaded a new version of File:Santos GH128 fig2.png

File:Santos GH128 fig2.png

2020-07-29T16:21:30Z

Camila Santos: Camila Santos uploaded a new version of File:Santos GH128 fig2.png

File:Santos GH128 fig2.png

2020-07-29T16:11:52Z

Camila Santos: Camila Santos uploaded a new version of File:Santos GH128 fig2.png

Glycoside Hydrolase Family 128

2020-07-28T02:51:24Z

Camila Santos:

{{UnderConstruction}}
* [[Author]]: ^^^Yuichi Sakamoto^^^ and ^^^Camilla Santos^^^
* [[Responsible Curator]]: ^^^Mario Murakami^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH128'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH128.html
|}
</div>


== Substrate specificities ==
Glycoside hydrolase Family 128 comprises prokaryotic and eukaryotic enzymes that are active on β-1,3-glucans. Endo-β-1,3-glucanases that degrade the carbohydrate at higher rates are found in bacterial subgroups (I and II) such as those from ''Amycolatopsis mediterranei'' <cite>Santos2020</cite> and ''Pseudomonas viridiflava'' <cite>Santos2020</cite>. Fungal enzymes, which are likely involved in cell wall remodeling processes, are more diverse in terms of activity: endo-β-1,3-glucanases, represented by the enzyme from ''Lentinula edodes'' (subgroup IV) <cite>Santos2020 Sakamoto2011</cite>; exo-β-1,3-glucanases that release trisaccharides (''Aureobsidium namibiae'', subgroup VI) <cite>Santos2020</cite> and monosaccharides (''Cryptococcus neoformans'', subgroup V) <cite>Santos2020</cite> from the reducing ends; and exo-β-1,3-glucanases that release trisaccharides from the non-reducing ends of triple-helical β-1,3-glucans, represented by the enzyme from ''Blastomyces gilchristii'' (subgroup III) <cite>Santos2020</cite>. Some fungal members from this family are devoid of catalytic activity but conserve the capacity to bind short β-1,3-glucooligosaccharides (subgroup VII) such as those from ''Trichoderma gamsii'' <cite>Santos2020</cite> and a second GH128 member from ''Cryptococcus neoformans'' <cite>Santos2020</cite>.

== Kinetics and Mechanism ==
Family 128 enzymes are retaining enzymes, which operate by a classical Koshland retention mechanism as confirmed through <sup>1</sup>H-nuclear magnetic resonance spectroscopy with the retention of the anomeric configuration of enzymatic products <cite>Santos2020</cite>.

== Catalytic Residues ==
As a clan GH-A family, the two acidic catalytic residues are located at the C-terminal ends of the strands β7 and β4. Both nucleophile and acid/base are glutamates and their function were validated by site-directed mutagenesis <cite>Santos2020</cite>

== Three-dimensional structures ==
The GH128 members exhibit a fold resembling an (α/β)<sub>8</sub>-barrel in which the helix α2 and the strand β3 are strictly absent <cite>Santos2020</cite>. Some enzymes such as the endo-β-1,3-glucanase from ''Lentinula edodes'' and the exo-β-1,3-glucanase from ''Cryptococcus neoformans'', also lack the helices α1 and α3, respectively <cite>Santos2020</cite>.

Two distinct modes of substrate binding were observed in the GH128 family <cite>Santos2020</cite>. The most widespread mode, named as hydrophobic knuckle, involves a tryptophan residue that interacts with four glucoside moieties from -5 to -2 and is fully complementary to the typically curved conformation of β-1,3-glucan chains. The other mode, only observed in fungal members belonging to subgroups IV and VI, requires substrate conformational changes to allow the binding to the catalytic interface. In these fungal subgroups, the hydrophobic knuckle is absent and two aromatic residues, positioned at the -5 and -4 subsites, create a linearized cleft, which requires a 180° torsion in the glycosidic bond between the glycosyl moieties -2 and -3 in the β-1,3-glucan chain for binding. This mode of substrate recognition is called as “flattening” mechanism due to the unusual conformational, but also stereochemically favorable, adopted by the substrate. It is notable that such mode of substrate binding was not yet observed in other CAZy families active on β-1,3-glucans.
== Clustering of GH128 ==

[[Image:Santos_GH128_fig2.png|thumb|right|250px|Figure 1. Clustering of the GH128 family into seven subgroups. Adapted from <cite>Santos2020</cite>.]]

After creation of the GH128 family by Y. Sakamoto and colleagues <cite>Sakamoto2011</cite>, the group headed by M. Murakami carried out a task force to explore the functional and structural diversity of this family <cite>Santos2020</cite>. For this purpose, they employed phylogenetic and SSN analyses to segregate the family into putative isofunctional subgroups. The SSN analysis resulted in two well discretized clusters (subgroups VI and VII) and a third cluster that was further subdivided into five subgroups (I to V) based on SSN alignment scores and evolutionary closeness (Fig. 1). Them, they characterized, biochemically and structurally, one or more members of each subgroup: AmGH128_I, PvGH128_II, ScGH128_II, BgGH128_III, LeGH128_IV, CnGH128_V, AnGH128_VI, TgGH128_VII and CnGH128_VII. Subgroups I and II were found to be predominantly present in bacteria, and the subgroups III to VII are mostly found in fungi. Bacterial enzymes are faster, present the hydrophobic knuckle and attack the β-1,3-glucan in an endo mode of action, which is compatible with their biological function: nutrition and competition. Fungal β-1,3-glucanases are known to act on remodeling of their own cell walls. Therefore, these enzymes are slower, more diverse in terms of substrate recognition modes (flattening mechanism - subgroups IV and VI; hydrophobic knuckle - subgroups III, V and VII) and mode of action (exo-enzymes - subgroups III, V and VI; endo-enzymes - subgroup IV; oligosaccharide binding protein - subgroup VII). It was the first time that a glycoside hydrolase family was rationally studied based on SSN analysis. It is noteworthy to mention that a recent study led by Prof. H. Brumer applied a similar strategy to classify the polyspecific GH16 family into isofunctional subgroups using the available functional and structural data in the literature <cite>Viborg2019</cite>. In addition, Brumer´s group made available an intuitive and robust program to perform SSN analyses, named as SSNpipe that is freely available from GitHub.
== Family Firsts ==
;First stereochemistry determination: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) by <sup>1</sup>H-NMR <cite>Santos2020</cite>.
;First catalytic nucleophile identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First general acid/base residue identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First 3-D structure: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.
== References ==
<biblio>
#Santos2020 pmid=32451508
#Sakamoto2011 pmid=21965406
#Viborg2019 pmid=31501245

</biblio>

[[Category:Glycoside Hydrolase Families|GH128]]

Glycoside Hydrolase Family 128

2020-07-28T02:51:01Z

Camila Santos:

{{UnderConstruction}}
* [[Author]]: ^^^Yuichi Sakamoto^^^ and ^^^Camilla Santos^^^
* [[Responsible Curator]]: ^^^Mario Murakami^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH128'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH128.html
|}
</div>


== Substrate specificities ==
Glycoside hydrolase Family 128 comprises prokaryotic and eukaryotic enzymes that are active on β-1,3-glucans. Endo-β-1,3-glucanases that degrade the carbohydrate at higher rates are found in bacterial subgroups (I and II) such as those from ''Amycolatopsis mediterranei'' <cite>Santos2020</cite> and ''Pseudomonas viridiflava'' <cite>Santos2020</cite>. Fungal enzymes, which are likely involved in cell wall remodeling processes, are more diverse in terms of activity: endo-β-1,3-glucanases, represented by the enzyme from ''Lentinula edodes'' (subgroup IV) <cite>Santos2020 Sakamoto2011</cite>; exo-β-1,3-glucanases that release trisaccharides (''Aureobsidium namibiae'', subgroup VI) <cite>Santos2020</cite> and monosaccharides (''Cryptococcus neoformans'', subgroup V) <cite>Santos2020</cite> from the reducing ends; and exo-β-1,3-glucanases that release trisaccharides from the non-reducing ends of triple-helical β-1,3-glucans, represented by the enzyme from ''Blastomyces gilchristii'' (subgroup III) <cite>Santos2020</cite>. Some fungal members from this family are devoid of catalytic activity but conserve the capacity to bind short β-1,3-glucooligosaccharides (subgroup VII) such as those from ''Trichoderma gamsii'' <cite>Santos2020</cite> and a second GH128 member from ''Cryptococcus neoformans'' <cite>Santos2020</cite>.

== Kinetics and Mechanism ==
Family 128 enzymes are retaining enzymes, which operate by a classical Koshland retention mechanism as confirmed through <sup>1</sup>H-nuclear magnetic resonance spectroscopy with the retention of the anomeric configuration of enzymatic products <cite>Santos2020</cite>.

== Catalytic Residues ==
As a clan GH-A family, the two acidic catalytic residues are located at the C-terminal ends of the strands β7 and β4. Both nucleophile and acid/base are glutamates and their function were validated by site-directed mutagenesis <cite>Santos2020</cite>

== Three-dimensional structures ==
The GH128 members exhibit a fold resembling an (α/β)<sub>8</sub>-barrel in which the helix α2 and the strand β3 are strictly absent <cite>Santos2020</cite>. Some enzymes such as the endo-β-1,3-glucanase from ''Lentinula edodes'' and the exo-β-1,3-glucanase from ''Cryptococcus neoformans'', also lack the helices α1 and α3, respectively <cite>Santos2020</cite>.

Two distinct modes of substrate binding were observed in the GH128 family <cite>Santos2020</cite>. The most widespread mode, named as hydrophobic knuckle, involves a tryptophan residue that interacts with four glucoside moieties from -5 to -2 and is fully complementary to the typically curved conformation of β-1,3-glucan chains. The other mode, only observed in fungal members belonging to subgroups IV and VI, requires substrate conformational changes to allow the binding to the catalytic interface. In these fungal subgroups, the hydrophobic knuckle is absent and two aromatic residues, positioned at the -5 and -4 subsites, create a linearized cleft, which requires a 180° torsion in the glycosidic bond between the glycosyl moieties -2 and -3 in the β-1,3-glucan chain for binding. This mode of substrate recognition is called as “flattening” mechanism due to the unusual conformational, but also stereochemically favorable, adopted by the substrate. It is notable that such mode of substrate binding was not yet observed in other CAZy families active on β-1,3-glucans.
== Clustering of GH128 ==

[[Image:Santos_GH128_fig2.png|thumb|right|250px|Figure 1. Clustering of the GH128 family into seven subgroups. Adapted from <cite>Santos2020</cite>.]].

After creation of the GH128 family by Y. Sakamoto and colleagues <cite>Sakamoto2011</cite>, the group headed by M. Murakami carried out a task force to explore the functional and structural diversity of this family <cite>Santos2020</cite>. For this purpose, they employed phylogenetic and SSN analyses to segregate the family into putative isofunctional subgroups. The SSN analysis resulted in two well discretized clusters (subgroups VI and VII) and a third cluster that was further subdivided into five subgroups (I to V) based on SSN alignment scores and evolutionary closeness (Fig. 1). Them, they characterized, biochemically and structurally, one or more members of each subgroup: AmGH128_I, PvGH128_II, ScGH128_II, BgGH128_III, LeGH128_IV, CnGH128_V, AnGH128_VI, TgGH128_VII and CnGH128_VII. Subgroups I and II were found to be predominantly present in bacteria, and the subgroups III to VII are mostly found in fungi. Bacterial enzymes are faster, present the hydrophobic knuckle and attack the β-1,3-glucan in an endo mode of action, which is compatible with their biological function: nutrition and competition. Fungal β-1,3-glucanases are known to act on remodeling of their own cell walls. Therefore, these enzymes are slower, more diverse in terms of substrate recognition modes (flattening mechanism - subgroups IV and VI; hydrophobic knuckle - subgroups III, V and VII) and mode of action (exo-enzymes - subgroups III, V and VI; endo-enzymes - subgroup IV; oligosaccharide binding protein - subgroup VII). It was the first time that a glycoside hydrolase family was rationally studied based on SSN analysis. It is noteworthy to mention that a recent study led by Prof. H. Brumer applied a similar strategy to classify the polyspecific GH16 family into isofunctional subgroups using the available functional and structural data in the literature <cite>Viborg2019</cite>. In addition, Brumer´s group made available an intuitive and robust program to perform SSN analyses, named as SSNpipe that is freely available from GitHub.
== Family Firsts ==
;First stereochemistry determination: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) by <sup>1</sup>H-NMR <cite>Santos2020</cite>.
;First catalytic nucleophile identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First general acid/base residue identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First 3-D structure: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.
== References ==
<biblio>
#Santos2020 pmid=32451508
#Sakamoto2011 pmid=21965406
#Viborg2019 pmid=31501245

</biblio>

[[Category:Glycoside Hydrolase Families|GH128]]

File:Santos GH128 fig2.png

2020-07-28T02:47:48Z

Camila Santos: Camila Santos uploaded a new version of File:Santos GH128 fig2.png

File:Santos GH128 fig2.png

2020-07-28T02:33:21Z

Camila Santos:

Glycoside Hydrolase Family 128

2020-07-24T03:01:49Z

Camila Santos:

{{UnderConstruction}}
* [[Author]]: ^^^Yuichi Sakamoto^^^ and ^^^Camilla Santos^^^
* [[Responsible Curator]]: ^^^Mario Murakami^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH128'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH128.html
|}
</div>


== Substrate specificities ==
Glycoside hydrolase Family 128 comprises prokaryotic and eukaryotic enzymes that are active on β-1,3-glucans. Endo-β-1,3-glucanases that degrade the carbohydrate at higher rates are found in bacterial subgroups (I and II) such as those from ''Amycolatopsis mediterranei'' <cite>Santos2020</cite> and ''Pseudomonas viridiflava'' <cite>Santos2020</cite>. Fungal enzymes, which are likely involved in cell wall remodeling processes, are more diverse in terms of activity: endo-β-1,3-glucanases, represented by the enzyme from ''Lentinula edodes'' (subgroup IV) <cite>Santos2020 Sakamoto2011</cite>; exo-β-1,3-glucanases that release trisaccharides (''Aureobsidium namibiae'', subgroup VI) <cite>Santos2020</cite> and monosaccharides (''Cryptococcus neoformans'', subgroup V) <cite>Santos2020</cite> from the reducing ends; and exo-β-1,3-glucanases that release trisaccharides from the non-reducing ends of triple-helical β-1,3-glucans, represented by the enzyme from ''Blastomyces gilchristii'' (subgroup III) <cite>Santos2020</cite>. Some fungal members from this family are devoid of catalytic activity but conserve the capacity to bind short β-1,3-glucooligosaccharides (subgroup VII) such as those from ''Trichoderma gamsii'' <cite>Santos2020</cite> and a second GH128 member from ''Cryptococcus neoformans'' <cite>Santos2020</cite>.

== Kinetics and Mechanism ==
Family 128 enzymes are retaining enzymes, which operate by a classical Koshland retention mechanism as confirmed through <sup>1</sup>H-nuclear magnetic resonance spectroscopy with the retention of the anomeric configuration of enzymatic products <cite>Santos2020</cite>.

== Catalytic Residues ==
As a clan GH-A family, the two acidic catalytic residues are located at the C-terminal ends of the strands β7 and β4. Both nucleophile and acid/base are glutamates and their function were validated by site-directed mutagenesis <cite>Santos2020</cite>

== Three-dimensional structures ==
The GH128 members exhibit a fold resembling an (α/β)<sub>8</sub>-barrel in which the helix α2 and the strand β3 are strictly absent <cite>Santos2020</cite>. Some enzymes such as the endo-β-1,3-glucanase from ''Lentinula edodes'' and the exo-β-1,3-glucanase from ''Cryptococcus neoformans'', also lack the helices α1 and α3, respectively <cite>Santos2020</cite>.

Two distinct modes of substrate binding were observed in the GH128 family <cite>Santos2020</cite>. The most widespread mode, named as hydrophobic knuckle, involves a tryptophan residue that interacts with four glucoside moieties from -5 to -2 and is fully complementary to the typically curved conformation of β-1,3-glucan chains. The other mode, only observed in fungal members belonging to subgroups IV and VI, requires substrate conformational changes to allow the binding to the catalytic interface. In these fungal subgroups, the hydrophobic knuckle is absent and two aromatic residues, positioned at the -5 and -4 subsites, create a linearized cleft, which requires a 180° torsion in the glycosidic bond between the glycosyl moieties -2 and -3 in the β-1,3-glucan chain for binding. This mode of substrate recognition is called as “flattening” mechanism due to the unusual conformational, but also stereochemically favorable, adopted by the substrate. It is notable that such mode of substrate binding was not yet observed in other CAZy families active on β-1,3-glucans.
== Clustering of GH128 ==

After creation of the GH128 family by Y. Sakamoto and colleagues <cite>Sakamoto2011</cite>, the group headed by M. Murakami carried out a task force to explore the functional and structural diversity of this family <cite>Santos2020</cite>. For this purpose, they employed phylogenetic and SSN analyses to segregate the family into putative isofunctional subgroups. The SSN analysis resulted in two well discretized clusters (subgroups VI and VII) and a third cluster that was further subdivided into five subgroups (I to V) based on SSN alignment scores and evolutionary closeness (Fig. 1). Them, they characterized, biochemically and structurally, one or more members of each subgroup: AmGH128_I, PvGH128_II, ScGH128_II, BgGH128_III, LeGH128_IV, CnGH128_V, AnGH128_VI, TgGH128_VII and CnGH128_VII. Subgroups I and II were found to be predominantly present in bacteria, and the subgroups III to VII are mostly found in fungi. Bacterial enzymes are faster, present the hydrophobic knuckle and attack the β-1,3-glucan in an endo mode of action, which is compatible with their biological function: nutrition and competition. Fungal β-1,3-glucanases are known to act on remodeling of their own cell walls. Therefore, these enzymes are slower, more diverse in terms of substrate recognition modes (flattening mechanism - subgroups IV and VI; hydrophobic knuckle - subgroups III, V and VII) and mode of action (exo-enzymes - subgroups III, V and VI; endo-enzymes - subgroup IV; oligosaccharide binding protein - subgroup VII). It was the first time that a glycoside hydrolase family was rationally studied based on SSN analysis. It is noteworthy to mention that a recent study led by Prof. H. Brumer applied a similar strategy to classify the polyspecific GH16 family into isofunctional subgroups using the available functional and structural data in the literature <cite>Viborg2019</cite>. In addition, Brumer´s group made available an intuitive and robust program to perform SSN analyses, named as SSNpipe that is freely available from GitHub.
== Family Firsts ==
;First stereochemistry determination: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) by <sup>1</sup>H-NMR <cite>Santos2020</cite>.
;First catalytic nucleophile identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First general acid/base residue identification: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First 3-D structure: ''Amycolatopsis mediterranei'' endo-β-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.
== References ==
<biblio>
#Santos2020 pmid=32451508
#Sakamoto2011 pmid=21965406
#Viborg2019 pmid=31501245

</biblio>

[[Category:Glycoside Hydrolase Families|GH128]]

User:Camila Santos

2020-06-22T03:30:29Z

Camila Santos:

[[Image:Camila_santos.jpg|200px|right]]

Camila Santos obtained her Ph.D. in Functional and Molecular Biology (with emphasis in Biochemistry) from the University of Campinas in 2009. Since then, she investigates the molecular mechanisms governing the action of CAZymes as researcher at the Brazilian Biorenewables National Laboratory. She has contributed to structural and functional studies of CAZymes from families [[GH2]] <cite>Domingues2018</cite>, [[GH5]] <cite>Santos2012a,Santos2012b,Alvarez2013a,Santos2015</cite>, [[GH10]] <cite>Santos2010,Alvarez2013b,Santos2014a</cite>, [[GH11]] <cite>Ribeiro2011</cite>, [[GH12]] <cite>Furtado2015</cite>, [[GH16]] <cite>Cota2011</cite>, [[GH39]] <cite>Santos2012c,Morais2020</cite>, [[GH43]] <cite>Santos2014b</cite>, [[GH51]] <cite>Souza2011,Santos2018</cite>, [[GH57]] <cite>Santos2011</cite> and [[GH128]] <cite>Santos2020</cite>. Her most recent contribution is on the family 128 <cite>Santos2020</cite>, which substantially expands the understanding of the molecular mechanisms for breakdown and modification of β-1,3-glucans.

----

'''References'''
<biblio>

#Domingues2018 pmid=29997257
#Santos2012a pmid=22155669
#Santos2012b pmid=21880019
#Alvarez2013a pmid=24358302
#Santos2015 pmid=25714929
#Santos2010 pmid=21070746
#Alvarez2013b pmid=23922891
#Santos2014a pmid=25266726
#Ribeiro2011 pmid=22006920
#Furtado2015 pmid=25605422
#Cota2011 pmid=21352806
#Santos2012c pmid=22993088
#Morais2020 pmid=32500063
#Santos2014b pmid=24469445
#Souza2011 pmid=21796714
#Santos2018 pmid=30127853
#Santos2011 pmid=21104698
#Santos2020 pmid=32451508

</biblio>


[[Category:Contributors|Santos,Camilla]]

User:Camila Santos

2020-06-22T03:29:30Z

Camila Santos:

[[Image:Camila_santos.png|200px|right]]

Camila Santos obtained her Ph.D. in Functional and Molecular Biology (with emphasis in Biochemistry) from the University of Campinas in 2009. Since then, she investigates the molecular mechanisms governing the action of CAZymes as researcher at the Brazilian Biorenewables National Laboratory. She has contributed to structural and functional studies of CAZymes from families [[GH2]] <cite>Domingues2018</cite>, [[GH5]] <cite>Santos2012a,Santos2012b,Alvarez2013a,Santos2015</cite>, [[GH10]] <cite>Santos2010,Alvarez2013b,Santos2014a</cite>, [[GH11]] <cite>Ribeiro2011</cite>, [[GH12]] <cite>Furtado2015</cite>, [[GH16]] <cite>Cota2011</cite>, [[GH39]] <cite>Santos2012c,Morais2020</cite>, [[GH43]] <cite>Santos2014b</cite>, [[GH51]] <cite>Souza2011,Santos2018</cite>, [[GH57]] <cite>Santos2011</cite> and [[GH128]] <cite>Santos2020</cite>. Her most recent contribution is on the family 128 <cite>Santos2020</cite>, which substantially expands the understanding of the molecular mechanisms for breakdown and modification of β-1,3-glucans.

----

'''References'''
<biblio>

#Domingues2018 pmid=29997257
#Santos2012a pmid=22155669
#Santos2012b pmid=21880019
#Alvarez2013a pmid=24358302
#Santos2015 pmid=25714929
#Santos2010 pmid=21070746
#Alvarez2013b pmid=23922891
#Santos2014a pmid=25266726
#Ribeiro2011 pmid=22006920
#Furtado2015 pmid=25605422
#Cota2011 pmid=21352806
#Santos2012c pmid=22993088
#Morais2020 pmid=32500063
#Santos2014b pmid=24469445
#Souza2011 pmid=21796714
#Santos2018 pmid=30127853
#Santos2011 pmid=21104698
#Santos2020 pmid=32451508

</biblio>


[[Category:Contributors|Santos,Camilla]]

File:Camila santos.jpg

2020-06-22T03:23:19Z

Camila Santos:

User:Camila Santos

2020-06-22T03:16:26Z

Camila Santos:

[[Image:Blank_user-200px.png|200px|right]]

Camila Santos obtained her Ph.D. in Functional and Molecular Biology (with emphasis in Biochemistry) from the University of Campinas in 2009. Since then, she investigates the molecular mechanisms governing the action of CAZymes as researcher at the Brazilian Biorenewables National Laboratory. She has contributed to structural and functional studies of CAZymes from families [[GH2]] <cite>Domingues2018</cite>, [[GH5]] <cite>Santos2012a,Santos2012b,Alvarez2013a,Santos2015</cite>, [[GH10]] <cite>Santos2010,Alvarez2013b,Santos2014a</cite>, [[GH11]] <cite>Ribeiro2011</cite>, [[GH12]] <cite>Furtado2015</cite>, [[GH16]] <cite>Cota2011</cite>, [[GH39]] <cite>Santos2012c,Morais2020</cite>, [[GH43]] <cite>Santos2014b</cite>, [[GH51]] <cite>Souza2011,Santos2018</cite>, [[GH57]] <cite>Santos2011</cite> and [[GH128]] <cite>Santos2020</cite>. Her most recent contribution is on the family 128 <cite>Santos2020</cite>, which substantially expands the understanding of the molecular mechanisms for breakdown and modification of β-1,3-glucans.

----

'''References'''
<biblio>

#Domingues2018 pmid=29997257
#Santos2012a pmid=22155669
#Santos2012b pmid=21880019
#Alvarez2013a pmid=24358302
#Santos2015 pmid=25714929
#Santos2010 pmid=21070746
#Alvarez2013b pmid=23922891
#Santos2014a pmid=25266726
#Ribeiro2011 pmid=22006920
#Furtado2015 pmid=25605422
#Cota2011 pmid=21352806
#Santos2012c pmid=22993088
#Morais2020 pmid=32500063
#Santos2014b pmid=24469445
#Souza2011 pmid=21796714
#Santos2018 pmid=30127853
#Santos2011 pmid=21104698
#Santos2020 pmid=32451508

</biblio>


[[Category:Contributors|Santos,Camilla]]

User:Camila Santos

2020-06-22T03:14:57Z

Camila Santos:

[[Image:Blank_user-200px.png|200px|right]]

Camila Santos obtained her Ph.D. in Functional and Molecular Biology (with emphasis in Biochemistry) from the University of Campinas in 2009. Since then, she investigates the molecular mechanisms governing the action of CAZymes as researcher at the Brazilian Biorenewables National Laboratory. She has contributed to structural and functional studies of CAZymes from families [[GH2]] <cite>Domingues2018</cite>, [[GH5]] <cite>Santos2012a,Santos2012b,Alvarez2013a,Santos2015</cite>, [[GH10]] <cite>Santos2010,Alvarez2013b,Santos2014a</cite>, [[GH11]] <cite>Ribeiro2011</cite>, [[GH12]] <cite>Furtado2015</cite>, [[GH16]] <cite>Cota2011</cite>, [[GH39]] <cite>Santos2012c,Morais2020</cite>, [[GH43]] <cite>Santos2014b</cite>, [[GH51]] <cite>Souza2011,Santos2018</cite>, [[GH57]] <cite>Santos2011</cite> and [[GH128]] <cite>Santos2020</cite>. Her most recent contribution is on the family 128 <cite>Santos2020</cite>, which substantially expands the understanding of the molecular mechanisms for breakdown and modification of β-1,3-glucans.

----

'''References'''
<biblio>

#Domingues2018 pmid=29997257
#Santos2012a pmid=22155669
#Santos2012b pmid=21880019
#Alvarez2013a pmid=24358302
#Santos2015 pmid=25714929
#Santos2010 pmid=21070746
#Alvarez2013b pmid=23922891
#Santos2014a pmid=25266726
#Ribeiro2011 pmid=22006920
#Furtado2015 pmid=25605422
#Cota2011 pmid=21352806
#Santos2012c pmid=22993088
#Morais2020 pmid=32500063
#Santos2014b pmid=24469445
#Souza2011 pmid=21796714
#Santos2018 pmid=30127853
#Santos2011 pmid=21104698
#Santos2020 pmid=32451508
</biblio>


[[Category:Contributors|Santos,Camilla]]