Glycoside Hydrolase Family 71

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• Author: Antonielle Vieira Monclaro
• Responsible Curator: Johan Larsbrink

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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

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 Trichoderma 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

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Catalytic Residues

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Three-dimensional structures

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Family Firsts

First stereochemistry determination

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First catalytic nucleophile identification

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First general acid/base residue identification

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First 3-D structure

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References

1. 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. DOI: 10.1016/0005-2744(72)90245-8

2. 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. DOI: 10.1080/00021369.1977.10862782

3. 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. DOI: 10.1074/jbc.275.3.2009

4. 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. DOI: 10.1371/journal.pone.0066853

5. 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. DOI: 10.1128/AEM.67.12.5833-5839.2001

6. 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. DOI: 10.1091/mbc.E04

7. 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. DOI: 10.1038/s42003-025-08696-3

8. 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. DOI: 10.1016/j.febslet.2006.05.062

9. 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. DOI: 10.1128/aem.00226-24

10. 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. DOI: 10.1016/j.bbrc.2025.151907