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Difference between revisions of "Glycoside Hydrolase Family 128"

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|retaining
 
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|'''Active site residues'''
 
|'''Active site residues'''
|predicted
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|known
 
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|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
 
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== Substrate specificities ==
 
== Substrate specificities ==
Family GH128 contains &beta;-1,3-glucanases that cleave &beta;-1,3 linkages in various &beta;-glucans such as lentinan from ''Lentinula edodes'', laminarin from ''Laminaria digitata'', pachyman from ''Poria cocos'', and curdlan from ''Alcaligenes faecalis''. The first GH128 enzyme, GLU1, was cloned from ''L. edodes'' fruiting bodies (shiitake mushroom). GLU1 did not degrade &beta;-1,3-linkages within &beta;-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>.
+
Glycoside hydrolase Family 128 comprises prokaryotic and eukaryotic enzymes that are active on &beta;-1,3-glucans. Endo-&beta;-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-&beta;-1,3-glucanases, represented by the enzyme from ''Lentinula edodes'' (subgroup IV) <cite>Santos2020 Sakamoto2011</cite>; exo-&beta;-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-&beta;-1,3-glucanases that release trisaccharides from the non-reducing ends of triple-helical &beta;-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 &beta;-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 ==
 
== Kinetics and Mechanism ==
''L. edodes'' GLU1 catalyzed the hydrolysis of glucans composed of &beta;-1,3-linked main chains, and reaction product analysis indicated that the enzyme had an endo-hydrolytic mode of action. The optimal pH and temperature of the enzyme for laminarin were pH 4 and at 50 °C, respectively. Detailed mechanistic studies on GH128 have not been performed, but three-dimensional structure prediction has indicated that GLU1 is a member of [[Clan]] GH-A. As such, GH128 members are predicted to be [[retaining]] enzymes <cite>Sakamoto2011</cite>.  
+
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 ==
 
== Catalytic Residues ==
From the sequence alignment of GH128 members, two conserved glutamic acids, E103 and E195 in ''L. edodes'' GLU1, were predicted to be the catalytic residues <cite>Sakamoto2011</cite>.
+
As a clan GH-A family, the two acidic catalytic residues are located at the C-terminal ends of the strands &beta;7 and &beta;4. Both nucleophile and acid/base are glutamates and their function were validated by site-directed mutagenesis <cite>Santos2020</cite>
  
 
== Three-dimensional structures ==
 
== Three-dimensional structures ==
A three-dimensional structure homology model of ''L. edodes'' GLU1 indicated similarity with several (beta/alpha)<sub>8</sub>-barrel (TIM-barrel) structures, including a [[GH39]] beta-xylosidase and a [[GH5]] beta-mannanase <cite>Sakamoto2011</cite>. Therefore, GH128 has been included in [[Clan]] [http://www.cazy.org/Glycoside-Hydrolases.html GH-A].
+
The GH128 members exhibit a fold resembling an (&alpha;/&beta;)<sub>8</sub>-barrel in which the helix &alpha;2 and the strand &beta;3 are strictly absent <cite>Santos2020</cite>. Some enzymes such as the endo-&beta;-1,3-glucanase from ''Lentinula edodes'' and the exo-&beta;-1,3-glucanase from ''Cryptococcus neoformans'', also lack the helices &alpha;1 and &alpha;3, 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 &beta;-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 &beta;-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 &beta;-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 &beta;-1,3-glucan in an endo mode of action, which is compatible with their biological function: nutrition and competition. Fungal &beta;-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 ==
 
== Family Firsts ==
;First stereochemistry determination: Predicted to be [[retaining]] by membership in [[Clan]] GH-A (not experimentally verified) <cite>Sakamoto2011</cite>.
+
;First stereochemistry determination: ''Amycolatopsis mediterranei'' endo-&beta;-1,3-glucanase (AmGH128_I) by <sup>1</sup>H-NMR <cite>Santos2020</cite>.
;First catalytic nucleophile identification: Predicted by sequence alignment (not experimentally verified) <cite>Sakamoto2011</cite>.
+
;First catalytic nucleophile identification: ''Amycolatopsis mediterranei'' endo-&beta;-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First general acid/base residue identification: Predicted by sequence alignment (not experimentally verified) <cite>Sakamoto2011</cite>.
+
;First general acid/base residue identification: ''Amycolatopsis mediterranei'' endo-&beta;-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis <cite>Santos2020</cite>.
;First 3-D structure: Predicted by modelling to be homologous to [[Clan]] GH-A (not experimentally verified) <cite>Sakamoto2011</cite>.
+
;First 3-D structure: ''Amycolatopsis mediterranei'' endo-&beta;-1,3-glucanase (AmGH128_I) <cite>Santos2020</cite>.
 
 
 
== References ==
 
== References ==
 
<biblio>
 
<biblio>
 +
#Santos2020 pmid=32451508
 
#Sakamoto2011 pmid=21965406
 
#Sakamoto2011 pmid=21965406
 +
#Viborg2019 pmid=31501245
  
 
</biblio>
 
</biblio>

Revision as of 20:01, 23 July 2020

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This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.

Glycoside Hydrolase Family GH128
Clan GH-A
Mechanism retaining
Active site residues known
CAZy DB link
http://www.cazy.org/GH128.html


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 [1] and Pseudomonas viridiflava [1]. 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) [1, 2]; exo-β-1,3-glucanases that release trisaccharides (Aureobsidium namibiae, subgroup VI) [1] and monosaccharides (Cryptococcus neoformans, subgroup V) [1] 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) [1]. 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 [1] and a second GH128 member from Cryptococcus neoformans [1].

Kinetics and Mechanism

Family 128 enzymes are retaining enzymes, which operate by a classical Koshland retention mechanism as confirmed through 1H-nuclear magnetic resonance spectroscopy with the retention of the anomeric configuration of enzymatic products [1].

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 [1]

Three-dimensional structures

The GH128 members exhibit a fold resembling an (α/β)8-barrel in which the helix α2 and the strand β3 are strictly absent [1]. 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 [1].

Two distinct modes of substrate binding were observed in the GH128 family [1]. 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 [2], the group headed by M. Murakami carried out a task force to explore the functional and structural diversity of this family [1]. 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 [3]. 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 1H-NMR [1].
First catalytic nucleophile identification
Amycolatopsis mediterranei endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis [1].
First general acid/base residue identification
Amycolatopsis mediterranei endo-β-1,3-glucanase (AmGH128_I), by site-directed mutagenesis based on structural analysis [1].
First 3-D structure
Amycolatopsis mediterranei endo-β-1,3-glucanase (AmGH128_I) [1].

References

  1. Santos CR, Costa PACR, Vieira PS, Gonzalez SET, Correa TLR, Lima EA, Mandelli F, Pirolla RAS, Domingues MN, Cabral L, Martins MP, Cordeiro RL, Junior AT, Souza BP, Prates ÉT, Gozzo FC, Persinoti GF, Skaf MS, and Murakami MT. (2020) Structural insights into β-1,3-glucan cleavage by a glycoside hydrolase family. Nat Chem Biol. 16, 920-929. DOI:10.1038/s41589-020-0554-5 | PubMed ID:32451508 | HubMed [Santos2020]
  2. Sakamoto Y, Nakade K, and Konno N. (2011) Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family. Appl Environ Microbiol. 77, 8350-4. DOI:10.1128/AEM.05581-11 | PubMed ID:21965406 | HubMed [Sakamoto2011]
  3. Viborg AH, Terrapon N, Lombard V, Michel G, Czjzek M, Henrissat B, and Brumer H. (2019) A subfamily roadmap of the evolutionarily diverse glycoside hydrolase family 16 (GH16). J Biol Chem. 294, 15973-15986. DOI:10.1074/jbc.RA119.010619 | PubMed ID:31501245 | HubMed [Viborg2019]
All Medline abstracts: PubMed | HubMed