CAZypedia - User contributions [en-ca]

User:Antonielle Vieira Monclaro

2026-01-29T10:04:20Z

Antonielle Vieira Monclaro:

[[File:AntonielleCMET.jpg|200px|right]]

I completed my PhD in 2018 at the University of Brasília (UnB), including a research secondment at the Norwegian University of Life Sciences (NMBU). I have worked as a scientific collaborator at EMBRAPA (Brazilian Agricultural Research Corporation), Université Libre de Bruxelles (ULB), Ghent University (UGent), and the Center for Plant Biotechnology and Genomics (CBGP).

I am currently a postdoctoral researcher at UGent and a collaborating researcher at UnB. My research focuses on CAZymes, mainly from filamentous fungi, including [[AA9]], [[GH5]], [[GH7]], [[GH11]], [[GH12]], [[GH45]], and others, targeting lignocellulosic biomass degradation and related biotechnological applications.

More information is available on my CMET profile: https://cmet.ugent.be/users/dr-antonielle-vieira-monclaro/

----


[[Category:Contributors|VieiraMonclaro,Antonielle]]

User:Antonielle Vieira Monclaro

2026-01-29T09:56:07Z

Antonielle Vieira Monclaro:

[[File:AntonielleCMET.jpg|200px|right]]

Antonielle completed her PhD in 2018 at the University of Brasília (UnB), with a research secondment at the Norwegian University of Life Sciences (NMBU). She has worked as a scientific collaborator at EMBRAPA (Brazilian Agricultural Research Corporation), at Université Libre de Bruxelles (ULB), at Ghent University (UGent), and at the Center for Plant Biotechnology and Genomics (CBGP).
She is currently a postdoctoral researcher at UGent, and a collaborating researcher at UnB. Her research focuses on carbohydrate-active enzymes (CAZymes), mainly from filamentous fungi, including [[AA9]], [[GH5]], [[GH7]], [[GH11]], [[GH12]], [[GH45]] and others, for lignocellulosic biomass degradation and biotechnological applications.

----


[[Category:Contributors|VieiraMonclaro,Antonielle]]

User:Antonielle Vieira Monclaro

2026-01-29T09:55:49Z

Antonielle Vieira Monclaro:

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

Antonielle completed her PhD in 2018 at the University of Brasília (UnB), with a research secondment at the Norwegian University of Life Sciences (NMBU). She has worked as a scientific collaborator at EMBRAPA (Brazilian Agricultural Research Corporation), at Université Libre de Bruxelles (ULB), at Ghent University (UGent), and at the Center for Plant Biotechnology and Genomics (CBGP).
She is currently a postdoctoral researcher at UGent, and a collaborating researcher at UnB. Her research focuses on carbohydrate-active enzymes (CAZymes), mainly from filamentous fungi, including [[AA9]], [[GH5]], [[GH7]], [[GH11]], [[GH12]], [[GH45]] and others, for lignocellulosic biomass degradation and biotechnological applications.

----


[[Category:Contributors|VieiraMonclaro,Antonielle]]

File:AntonielleCMET.jpg

2026-01-29T09:53:37Z

Antonielle Vieira Monclaro:

Glycoside Hydrolase Family 71

2026-01-22T12:38:57Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''T. harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''A. nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>.

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''S. pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''A. nidulans'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''S. pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''A. nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:35:51Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''A. nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>.

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''S. pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''A. nidulans'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''S. pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''A. nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:34:51Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''A. nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>.

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''S. pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''A. nidulans'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:33:23Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''A. nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>.

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''S. pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:32:52Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''A. nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>.

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:32:29Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''A. nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:29:30Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan (from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:08:06Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan ((from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 pmid=15194814

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:02:38Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan ((from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K. (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric Biol Chem''. 1977;'''41'''(10);1889-95. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 Dekker, N., Speijer, D., Grün, C.H., Van den Berg, M., De Haan, A. and Hochstenbach, F. (2004) ‘Role of the α-glucanase Agn1p in fission-yeast cell separation’, Molecular Biology of the Cell, 15, pp. 3903–3914. [https://doi.org/10.1091/mbc.E04 DOI: 10.1091/mbc.E04]

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T12:01:04Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan ((from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai K, Kobayashi M, Matsuda K (1977). ''Properties of an α-1,3-glucanase from Streptomyces sp. KI-8''. ''Agric. Biol. Chem''. 1977;'''41'''(10);1889-1895. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904

#VillalobosDuno2013 pmid=23825576
#AitLahsen2001 pmid=11722942

#Dekker2004 Dekker, N., Speijer, D., Grün, C.H., Van den Berg, M., De Haan, A. and Hochstenbach, F. (2004) ‘Role of the α-glucanase Agn1p in fission-yeast cell separation’, Molecular Biology of the Cell, 15, pp. 3903–3914. [https://doi.org/10.1091/mbc.E04 DOI: 10.1091/mbc.E04]

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-22T11:53:52Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>

[[File: AnGH71C.png|thumb|right|500px|'''Figure 1. Structure of ''An''GH71C from ''Aspergillus nidulans''.''' The structure solved with nigerooligosaccharides as ligand is shown (PDB ID [{{PDBlink}}9fnh 9FNH]), with one protein molecule and oligosaccharides from different chains that together span the active site, as blue sticks. The core catalytic (α/β)8 barrel is shown in silver, the C-terminal β-sheet domain in pale orange, and the catalytic residues in green.]]

== Substrate specificities ==

GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan ((from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 pmid=4622000

#Imai1977 Imai, K., Kobayashi, M. and Matsuda, K. (1977) ‘Properties of an α-1,3-glucanase from Streptomyces sp. KI-8’, Agricultural and Biological Chemistry, 41, pp. 1889–1895. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 pmid=10636904
#VillalobosDuno2013 pmid=23825576

#AitLahsen2001 pmid=11722942

#Dekker2004 Dekker, N., Speijer, D., Grün, C.H., Van den Berg, M., De Haan, A. and Hochstenbach, F. (2004) ‘Role of the α-glucanase Agn1p in fission-yeast cell separation’, Molecular Biology of the Cell, 15, pp. 3903–3914. [https://doi.org/10.1091/mbc.E04 DOI: 10.1091/mbc.E04]

#Mazurkewich2025 pmid=40877455

#Grun2006 pmid=16780840

#Sinitsyna2025 pmid=39846749

#Horaguchi2025 pmid=40306164
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

User:Antonielle Vieira Monclaro

2026-01-13T16:29:30Z

Antonielle Vieira Monclaro:

[[Image:Blank_user-200px.png|200px|right]]
Antonielle completed her PhD in 2018 at the University of Brasília (UnB), with a research secondment at the Norwegian University of Life Sciences (NMBU). She has worked as a scientific collaborator at EMBRAPA (Brazilian Agricultural Research Corporation), at Université Libre de Bruxelles (ULB), at Ghent University (UGent), and at the Center for Plant Biotechnology and Genomics (CBGP).

She is currently a postdoctoral researcher at UGent, and a collaborating researcher at UnB. Her research focuses on carbohydrate-active enzymes (CAZymes), mainly from filamentous fungi, including AA9, GH5, GH7, GH11, GH12, GH45 and others, for lignocellulosic biomass degradation and biotechnological applications.

----


[[Category:Contributors|VieiraMonclaro,Antonielle]]

User:Antonielle Vieira Monclaro

2026-01-13T16:28:39Z

Antonielle Vieira Monclaro:

[[Image:Blank_user-200px.png|200px|right]]
Antonielle completed her PhD in 2018 at the University of Brasília (UnB), with a research secondment at the Norwegian University of Life Sciences (NMBU). She has worked as a scientific collaborator at EMBRAPA (Brazilian Agricultural Research Corporation), at Université Libre de Bruxelles (ULB), at Ghent University (UGent), and at the Center for Plant Biotechnology and Genomics (CBGP, Spain).

She is currently a postdoctoral researcher at UGent, and a collaborating researcher at UnB. Her research focuses on carbohydrate-active enzymes (CAZymes), mainly from filamentous fungi, including AA9, GH5, GH7, GH11, GH12, GH45 and others, for lignocellulosic biomass degradation and biotechnological applications.

----

<biblio>

</biblio>


[[Category:Contributors|VieiraMonclaro,Antonielle]]

Glycoside Hydrolase Family 71

2026-01-13T16:19:11Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>


== Substrate specificities ==
GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan ((from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: The stereochemistry of GH71 enzymes has been resolved by monitoring the anomeric configuration of the released glucose using ¹H NMR spectroscopy, confirming that the enzymes operate through the inversion mechanism <cite>Grun2006</cite>.
;First catalytic nucleophile identification: Content is to be added here.
;First general acid/base residue identification: In AnGH71C, the catalytic residues have been identified as a dyad, with an aspartate residue (Asp265) acting as a general base that activates the catalytic water molecule, and a glutamate residue (Glu268) acting as a general acid that protonates the leaving group <cite>Mazurkewich2025</cite>.
;First 3-D structure: The first solved structure of a GH71 enzyme was of Agn1p from ''Schizosaccharomyces pombe'', published in June 2025 by Horaguchi et al. <cite>Horaguchi2025</cite>, which demonstrated that members of the GH71 family possess a classic (β/α)₈ TIM-barrel core closely associated with a C-terminal β-sandwich accessory domain. In August the same year, Mazurkewich et al. published the structure of AnGH71C from ''Aspergillus nidulans'', which additionally included structures with glucose and nigerotetraose bound in the active site, respectively <cite>Mazurkewich2025</cite>.

== References ==
<biblio>
#Zonneveld1972 Zonneveld, B.J.M. (1972) ‘A new type of enzyme, an exo-splitting α-1,3 glucanase from non-induced cultures of Aspergillus nidulans’, Biochimica et Biophysica Acta (BBA) – Enzymology, 258, pp. 541–547. [https://doi.org/10.1016/0005-2744(72)90245-8 DOI: 10.1016/0005-2744(72)90245-8]

#Imai1977 Imai, K., Kobayashi, M. and Matsuda, K. (1977) ‘Properties of an α-1,3-glucanase from Streptomyces sp. KI-8’, Agricultural and Biological Chemistry, 41, pp. 1889–1895. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 Fuglsang, C.C., Berka, R.M., Wahleithner, J.A., Kauppinen, S., Shuster, J.R., Rasmussen, G., Halkier, T., Dalbøge, H. and Henrissat, B. (2000) ‘Biochemical analysis of recombinant fungal mutanases’, Journal of Biological Chemistry, 275, pp. 2009–2018. [https://doi.org/10.1074/jbc.275.3.2009 DOI: 10.1074/jbc.275.3.2009]

#VillalobosDuno2013 Villalobos-Duno, H., San-Blas, G., Paulinkevicius, M., Sánchez-Martín, Y. and Nino-Vega, G. (2013) ‘Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous expression in Schizosaccharomyces pombe’, PLoS ONE, 8, e66853. [https://doi.org/10.1371/journal.pone.0066853 DOI: 10.1371/journal.pone.0066853]

#AitLahsen2001 Ait-Lahsen, H., Soler, A., Rey, M., De La Cruz, J., Monte, E. and Llobell, A. (2001) ‘An antifungal exo-α-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum’, Applied and Environmental Microbiology, 67, pp. 5833–5839. [https://doi.org/10.1128/AEM.67.12.5833-5839.2001 DOI: 10.1128/AEM.67.12.5833-5839.2001]

#Dekker2004 Dekker, N., Speijer, D., Grün, C.H., Van den Berg, M., De Haan, A. and Hochstenbach, F. (2004) ‘Role of the α-glucanase Agn1p in fission-yeast cell separation’, Molecular Biology of the Cell, 15, pp. 3903–3914. [https://doi.org/10.1091/mbc.E04 DOI: 10.1091/mbc.E04]

#Mazurkewich2025 Mazurkewich, S., Widén, T., Karlsson, H., Evenäs, L., Ramamohan, P., Wohlert, J., Brändén, G. and Larsbrink, J. (2025) ‘Structural and biochemical basis for activity of Aspergillus nidulans α-1,3-glucanases from glycoside hydrolase family 71’, Communications Biology, 8. [https://doi.org/10.1038/s42003-025-08696-3 DOI: 10.1038/s42003-025-08696-3]

#Grun2006 Grün, C.H., Dekker, N., Nieuwland, A.A., Klis, F.M., Kamerling, J.P., Vliegenthart, J.F.G. and Hochstenbach, F. (2006) ‘Mechanism of action of the endo-(1→3)-α-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum’, FEBS Letters, 580, pp. 3780–3786. [https://doi.org/10.1016/j.febslet.2006.05.062 DOI: 10.1016/j.febslet.2006.05.062]

#Sinitsyna2025 Sinitsyna, O.A., Volkov, P.V., Zorov, I.N., Rozhkova, A.M., Emshanov, O.V., Romanova, Y.M., Komarova, B.S., Novikova, N.S., Nifantiev, N.E. and Sinitsyn, A.P. (2025) ‘Physico-chemical properties and substrate specificity of α-(1→3)-D-glucan degrading recombinant mutanase from Trichoderma harzianum expressed in Penicillium verruculosum’, Applied and Environmental Microbiology, 91. [https://doi.org/10.1128/aem.00226-24 DOI: 10.1128/aem.00226-24]

#Horaguchi2025 Horaguchi, Y., Saitoh, H., Konno, H., Makabe, K. and Yano, S. (2025) ‘Crystal structure of GH71 α-1,3-glucanase Agn1p from Schizosaccharomyces pombe: an enzyme regulating cell division in fission yeast’, Biochemical and Biophysical Research Communications, 766. [https://doi.org/10.1016/j.bbrc.2025.151907 DOI: 10.1016/j.bbrc.2025.151907]
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-13T16:17:40Z

Antonielle Vieira Monclaro:

{{UnderConstruction}}
* [[Author]]: [[User:Antonielle Vieira Monclaro|Antonielle Vieira Monclaro]]
* [[Responsible Curator]]: [[User:Johan Larsbrink|Johan Larsbrink]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH71'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH71.html
|}
</div>


== Substrate specificities ==
GH71 comprises enzymes with α-1,3-glucanase activity (EC 3.2.1.59), often referred to as mutanases, based on mutan being an alternative name for α-1,3-glucan ((from ''Streptococcus mutans''). Early studies demonstrated that these enzymes hydrolyze pure α-1,3-glucans while remaining inactive toward α-glucans containing mixed α-1,3/α-1,4 linkages <cite>Zonneveld1972</cite>. Subsequent work showed that GH71 enzymes act on a broader range of α-1,3-linked glucans, including pseudonigeran and soluble carboxymethylated α-1,3-glucan, but display no activity toward other tested α- or β-linked glycans <cite>Imai1977 Fuglsang2000 VillalobosDuno2013 AitLahsen2001 Dekker2004 Mazurkewich2025</cite>.

Depending on the enzyme, GH71 α-1,3-glucanases may exhibit exo- or endo-type hydrolytic activity. Some enzymes with exo activity, such as Agn13.1 from ''Trichoderma harzianum'', showed a 1:1 correlation between glucose released and reducing sugars, typical of exo hydrolysis, and was unable to cleave periodate-oxidized S-glucan, which is resistant to exo-α-1,3-glucanases <cite>AitLahsen2001</cite>. Endo-acting GH71 enzymes include Agn1p from ''Schizosaccharomyces pombe'' which does not hydrolyze pNP-α-glucose, and is not inhibited by classical exo-glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, or D-glucono-1,5-lactone <cite>Dekker2004</cite>. MutAp from ''Trichoderma harzianum'', an endo-hydrolytic α-1,3-glucanase, is suggested to act processively from the non-reducing end, repeatedly releasing glucose before dissociating <cite>Grun2006 Sinitsyna2025</cite>. Its insensitivity to multiple exo-glycosidase inhibitors, and experiments with reduced oligosaccharides (e.g., G5-ol) further yield no products compatible with exo activity (e.g., G4-ol). The minimum chain-length requirement for MutAp has been shown to be a tetrasaccharide.

The ''Aspergillus nidulans'' enzymes AnGH71B and AnGH71C display distinct behaviors when acting on reduced oligosaccharides (nigeropentaose and nigerohexaose), reflecting different cleavage mechanisms <cite>Mazurkewich2025</cite>. AnGH71C exhibits a pattern consistent with endo-cleavage, evidenced by the diverse products generated from reduced nigerohexaose. In contrast, AnGH71B displays exo-processive characteristics despite the absence of released reduced glucose, explained by the inability of subsite +1 to accommodate the reduced unit and therefore preventing classical terminal cleavage.

Overall, GH71 enzymes exhibit strict specificity for continuous regions of α-1,3-glycosidic linkages, with no tolerance for alternating segments containing α-1,4 linkages <cite>Zonneveld1972 AitLahsen2001</cite>, as found in the polysaccharide nigeran (α-1,3/1,4-glucan). End products range from glucose (e.g. from endo-acting processive action), to nigerooligosaccharides with DP 2–7 <cite>VillalobosDuno2013 Dekker2004 Sinitsyna2025</cite>. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes <cite>Mazurkewich2025 Grun2006</cite>.

== Kinetics and Mechanism ==
The anomeric configuration of the products released by α-1,3-glucanases has been elucidated by complementary NMR and crystallography approaches. In the case of MutAp from ''Trichoderma harzianum'', the hydrolysis of carboxymethylated α-1,3-glucan was monitored by ¹H NMR, revealing the appearance of β-Glc signals and the complete absence of α-Glc, demonstrating inversion of the anomeric configuration <cite>Grun2006</cite>. NMR studies of AnGH71B and AnGH71C from ''Aspergillus nidulans'' likewise showed inversion of products, and structures including the inverted product nigerose further supports these findings <cite>Mazurkewich2025</cite>

== Catalytic Residues ==
Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. <cite>Horaguchi2025</cite>. Individual substitutions of these residues (D69N, D237A/N, E240A/Q) led to drastic reductions in activity on α-1,3-glucan.

Structure-guided mutational studies of AnGH71B and AnGH71C directly identified the catalytic residues Asp265 (general base) and Glu268 (general acid), functionally separating subsites −4 to +3 of the enzymes <cite>Mazurkewich2025</cite>. The simultaneous observation of the α-linked substrate nigerotetraose and β-anomer of nigerotriose as product in the active site, together with an arrangement of a water molecule positioned ~3.2 Å from the anomeric carbon of the substrate, supported a classic inverting mechanism, in which Asp265 activates the nucleophilic water and Glu268 protonates the leaving group. Substitution of these residues resulted in 200- to 15,000-fold reductions in activity, confirming their catalytic role.

== Three-dimensional structures ==
The three-dimensional structure of GH71 enzymes has been elucidated through two independent crystallographic studies, both revealing that members of this family adopt a classic (β/α)₈ TIM-barrel core, closely associated with a C-terminal β-sandwich accessory domain <cite>Horaguchi2025 Mazurkewich2025</cite>.

The first structural description, obtained for ''Schizosaccharomyces pombe'' Agn1p, showed that its TIM barrel forms a deep cavity accessible to the solvent, consistent with the catalytic cleft observed in other glycoside hydrolases. Structural work on ''Aspergillus niger'' AnGH71C corroborated this overall fold and showed that the β-sandwich closely resembles an Ig-like fibronectin III domain, compacting closely against the TIM barrel to form a long substrate-binding cleft comprising at least seven subsites (−4 to +3). The structures of ligand complexes revealed minimal protein rearrangement upon binding but highlighted a conformational packing of the β6–α6 loop over subsites +1 to +3, contributing to substrate stabilization.

Simulations and geometries of the bound state further indicated that GH71 enzymes exploit the intrinsic low-energy conformations of α-1,3-linked oligosaccharides, while a high-energy configuration around the −1/+1 region likely prepares the glycosidic bond for cleavage.

== Family Firsts ==
;First stereochemistry determination: Content is to be added here.
;First catalytic nucleophile identification: Content is to be added here.
;First general acid/base residue identification: Content is to be added here.
;First 3-D structure: Content is to be added here.

== References ==
<biblio>
#Zonneveld1972 Zonneveld, B.J.M. (1972) ‘A new type of enzyme, an exo-splitting α-1,3 glucanase from non-induced cultures of Aspergillus nidulans’, Biochimica et Biophysica Acta (BBA) – Enzymology, 258, pp. 541–547. [https://doi.org/10.1016/0005-2744(72)90245-8 DOI: 10.1016/0005-2744(72)90245-8]

#Imai1977 Imai, K., Kobayashi, M. and Matsuda, K. (1977) ‘Properties of an α-1,3-glucanase from Streptomyces sp. KI-8’, Agricultural and Biological Chemistry, 41, pp. 1889–1895. [https://doi.org/10.1080/00021369.1977.10862782 DOI: 10.1080/00021369.1977.10862782]

#Fuglsang2000 Fuglsang, C.C., Berka, R.M., Wahleithner, J.A., Kauppinen, S., Shuster, J.R., Rasmussen, G., Halkier, T., Dalbøge, H. and Henrissat, B. (2000) ‘Biochemical analysis of recombinant fungal mutanases’, Journal of Biological Chemistry, 275, pp. 2009–2018. [https://doi.org/10.1074/jbc.275.3.2009 DOI: 10.1074/jbc.275.3.2009]

#VillalobosDuno2013 Villalobos-Duno, H., San-Blas, G., Paulinkevicius, M., Sánchez-Martín, Y. and Nino-Vega, G. (2013) ‘Biochemical characterization of Paracoccidioides brasiliensis α-1,3-glucanase Agn1p, and its functionality by heterologous expression in Schizosaccharomyces pombe’, PLoS ONE, 8, e66853. [https://doi.org/10.1371/journal.pone.0066853 DOI: 10.1371/journal.pone.0066853]

#AitLahsen2001 Ait-Lahsen, H., Soler, A., Rey, M., De La Cruz, J., Monte, E. and Llobell, A. (2001) ‘An antifungal exo-α-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum’, Applied and Environmental Microbiology, 67, pp. 5833–5839. [https://doi.org/10.1128/AEM.67.12.5833-5839.2001 DOI: 10.1128/AEM.67.12.5833-5839.2001]

#Dekker2004 Dekker, N., Speijer, D., Grün, C.H., Van den Berg, M., De Haan, A. and Hochstenbach, F. (2004) ‘Role of the α-glucanase Agn1p in fission-yeast cell separation’, Molecular Biology of the Cell, 15, pp. 3903–3914. [https://doi.org/10.1091/mbc.E04 DOI: 10.1091/mbc.E04]

#Mazurkewich2025 Mazurkewich, S., Widén, T., Karlsson, H., Evenäs, L., Ramamohan, P., Wohlert, J., Brändén, G. and Larsbrink, J. (2025) ‘Structural and biochemical basis for activity of Aspergillus nidulans α-1,3-glucanases from glycoside hydrolase family 71’, Communications Biology, 8. [https://doi.org/10.1038/s42003-025-08696-3 DOI: 10.1038/s42003-025-08696-3]

#Grun2006 Grün, C.H., Dekker, N., Nieuwland, A.A., Klis, F.M., Kamerling, J.P., Vliegenthart, J.F.G. and Hochstenbach, F. (2006) ‘Mechanism of action of the endo-(1→3)-α-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum’, FEBS Letters, 580, pp. 3780–3786. [https://doi.org/10.1016/j.febslet.2006.05.062 DOI: 10.1016/j.febslet.2006.05.062]

#Sinitsyna2025 Sinitsyna, O.A., Volkov, P.V., Zorov, I.N., Rozhkova, A.M., Emshanov, O.V., Romanova, Y.M., Komarova, B.S., Novikova, N.S., Nifantiev, N.E. and Sinitsyn, A.P. (2025) ‘Physico-chemical properties and substrate specificity of α-(1→3)-D-glucan degrading recombinant mutanase from Trichoderma harzianum expressed in Penicillium verruculosum’, Applied and Environmental Microbiology, 91. [https://doi.org/10.1128/aem.00226-24 DOI: 10.1128/aem.00226-24]

#Horaguchi2025 Horaguchi, Y., Saitoh, H., Konno, H., Makabe, K. and Yano, S. (2025) ‘Crystal structure of GH71 α-1,3-glucanase Agn1p from Schizosaccharomyces pombe: an enzyme regulating cell division in fission yeast’, Biochemical and Biophysical Research Communications, 766. [https://doi.org/10.1016/j.bbrc.2025.151907 DOI: 10.1016/j.bbrc.2025.151907]
</biblio>


[[Category:Glycoside Hydrolase Families|GH071]]

Glycoside Hydrolase Family 71

2026-01-13T16:16:46Z

Antonielle Vieira Monclaro:

Glycoside Hydrolase Family 71

2026-01-13T16:16:18Z

Antonielle Vieira Monclaro:

Glycoside Hydrolase Family 71

2026-01-13T16:15:07Z

Antonielle Vieira Monclaro: