Glycoside Hydrolase Family 71

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Glycoside Hydrolase Family GH71
Clan	GH-x
Mechanism	inverting
Active site residues	known
CAZy DB link
https://www.cazy.org/GH71.html

Figure 1. Structure of AnGH71C from Aspergillus nidulans. The structure solved with nigerooligosaccharides as ligand is shown (PDB ID 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 [1]. 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 [2, 3, 4, 5, 6, 7].

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 [5]. 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 [6]. 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 [8, 9]. 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 [7]. 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 [1, 5], 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 [4, 6, 9]. Nigerotriose has been found as a final product together with glucose from endo-acting processive GH71 enzymes [7, 8].

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 T. 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 [8]. NMR studies of AnGH71B and AnGH71C from A. nidulans likewise showed inversion of products, and structures including the inverted anomer of the product nigerose further supports these findings [7].

Catalytic Residues

Three conserved acidic residues (Asp69, Asp237, and Glu240) were identified in the active site of Agn1p by Horaguchi et al. [10]. 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 [7]. 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 [7, 10].

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 [8].
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 [7].
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. [10], 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 nigerooligosaccharides bound in the active site, respectively [7].

References

All Medline abstracts: PubMed