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Structural insights into β-1,3-glucan cleavage by a glycoside hydrolase family

A Publisher Correction to this article was published on 25 June 2020

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Abstract

The fundamental and assorted roles of β-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on β-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/β)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical β-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of β-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of β-1,3-glucans, which can be exploited for biotechnological applications.

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Fig. 1: Clustering and phylogeny of the GH128 family.
Fig. 2: Structure, substrate recognition and bacterial subgroups.
Fig. 3: Structural determinants for β-1,3-glucan binding in subgroups III, V and VII.
Fig. 4: The unconventional substrate-binding mode of subgroups IV and VI.
Fig. 5: β-1,3-glucan recognition and cleavage by the GH128 family.

Data availability

Structural data have been deposited in the Protein Data Bank (https://www.rcsb.org/) under accession codes 6UAQ (AmGH128_I), 6UAR (AmGH128_I, L3), 6UAS (AmGH128_I/E199A, L5+GLC), 6UFL (AmGH128_I/E199Q, L6), 6UFZ (AmGH128_I/E199Q), 6UAT (AmGH128_I/E102A, L5), 6UAU (AmGH128_I/E102A, L3 + L2), 6UAV (PvGH128_II), 6UAW (PvGH128_II, L3), 6UAX (ScGH128_II), 6UAY (BgGH128_III), 6UAZ (BgGH128_III, GLC), 6UB0 (BgGH128_III, L2), 6UB1 (BgGH128_III, L3), 6UB2 (LeGH128_IV), 6UB3 (LeGH128_IV, L2), 6UB4 (LeGH128_IV, L3(C2)), 6UB5 (LeGH128_IV, L3 (P21)), 6UB6 (LeGH128_IV, L4), 6UB7 (CnGH128_V), 6UB8 (AnGH128_VI), 6UBA (AnGH128_VI, L3), 6UBB (AnGH128_VI, exo-site), 6UBC (CnGH128_VII) and 6UBD (TgGH128_VII). All other data generated or analyzed during this study are included in this published article (and its Supplementary information files) or are available from the corresponding author on reasonable request.

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References

  1. Stone, B. A. in Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides (eds Bacic, A., Fincher, G. B. & Stone, B. A.) 5–46 (Academic Press, 2009).

  2. Gidley, M. J. & Nishinari, K. in Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides 47–118 (Academic Press, 2009).

  3. McIntosh, M., Stone, B. A. & Stanisich, V. A. Curdlan and other bacterial (1–>3)-beta-d-glucans. Appl. Microbiol. Biotechnol. 68, 163–173 (2005).

    CAS  PubMed  Article  Google Scholar 

  4. Kang, X. et al. Molecular architecture of fungal cell walls revealed by solid-state NMR. Nat. Commun. 9, 2747 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  5. Helbert, W. et al. Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space. Proc. Natl Acad. Sci. USA 116, 6063–6068 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Ishida, T. et al. Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Bianchetti, C. M. et al. Active site and laminarin binding in glycoside hydrolase family 55. J. Biol. Chem. 290, 11819–11832 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Papageorgiou, A. C., Chen, J. & Li, D. Crystal structure and biological implications of a glycoside hydrolase family 55 beta-1,3-glucanase from Chaetomium thermophilum. Biochim. Biophys. Acta Proteins Proteom. 1865, 1030–1038 (2017).

    CAS  PubMed  Article  Google Scholar 

  9. Wu, H. M. et al. Structure, mechanistic action, and essential residues of a GH-64 enzyme, laminaripentaose-producing beta-1,3-glucanase. J. Biol. Chem. 284, 26708–26715 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Qin, Z. et al. The recognition mechanism of triple-helical β-1,3-glucan by a β-1,3-glucanase. Chem. Commun. 53, 9368–9371 (2017).

    CAS  Article  Google Scholar 

  11. Zhou, P. et al. The structure of a glycoside hydrolase family 81 endo-beta-1,3-glucanase. Acta Crystallogr. D. 69, 2027–2038 (2013).

    CAS  PubMed  Article  Google Scholar 

  12. Pluvinage, B., Fillo, A., Massel, P. & Boraston, A. B. Structural analysis of a family 81 glycoside hydrolase implicates its recognition of beta-1,3-glucan quaternary. Structure 25, 1348–1359 e3 (2017).

    CAS  PubMed  Article  Google Scholar 

  13. Sakamoto, Y., Nakade, K. & Konno, N. Endo-beta-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family. Appl. Environ. Microbiol. 77, 8350–8354 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Masuda, T. et al. Subatomic structure of hyper-sweet thaumatin D21N mutant reveals the importance of flexible conformations for enhanced sweetness. Biochimie 157, 57–63 (2019).

    CAS  PubMed  Article  Google Scholar 

  15. Terrapon, N. et al. PULDB: the expanded database of polysaccharide utilization loci. Nucleic Acids Res. 46, D677–D683 (2018).

    CAS  PubMed  Article  Google Scholar 

  16. Boraston, A. B., Warren, R. A. & Kilburn, D. G. Beta-1,3-glucan binding by a thermostable carbohydrate-binding module from thermotoga maritima. Biochemistry 40, 14679–14685 (2001).

    CAS  PubMed  Article  Google Scholar 

  17. van Bueren, A. L., Morland, C., Gilbert, H. J. & Boraston, A. B. Family 6 carbohydrate binding modules recognize the non-reducing end of beta-1,3-linked glucans by presenting a unique ligand binding surface. J. Biol. Chem. 280, 530–537 (2005).

    PubMed  Article  CAS  Google Scholar 

  18. Jam, M. et al. Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module with its native laminarin ligand. FEBS J. 283, 1863–1879 (2016).

    CAS  PubMed  Article  Google Scholar 

  19. Brunecky, R. et al. Revealing nature’s cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science 342, 1513–1516 (2013).

    CAS  PubMed  Article  Google Scholar 

  20. Henrissat, B. et al. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc. Natl Acad. Sci. USA 92, 7090–7094 (1995).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Koshland, D. E. Jr Stereochemistry and the mechanism of enzymatic reactions. Bio. Rev. 28, 416–436 (1953).

    CAS  Article  Google Scholar 

  22. Davies, G. & Henrissat, B. Structures and mechanisms of glycosyl hydrolases. Structure 3, 853–859 (1995).

    CAS  PubMed  Article  Google Scholar 

  23. Kim, H. W. & Ishikawa, K. Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots. Biochem. J. 437, 223–230 (2011).

    CAS  PubMed  Article  Google Scholar 

  24. Planas, A. Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering. Biochim. Biophys. Acta 1543, 361–382 (2000).

    CAS  PubMed  Article  Google Scholar 

  25. Gloster, T. M. et al. Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12. J. Biol. Chem. 282, 19177–19189 (2007).

    CAS  PubMed  Article  Google Scholar 

  26. Gueguen, Y., Voorhorst, W. G., van der Oost, J. & de Vos, W. M. Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic Archaeon pyrococcus furiosus. J. Biol. Chem. 272, 31258–31264 (1997).

    CAS  PubMed  Article  Google Scholar 

  27. Kumagai, Y. & Ojima, T. Isolation and characterization of two types of beta-1,3-glucanases from the common sea hare Aplysia kurodai. Comp. Biochem. Physiol. B. 155, 138–144 (2010).

    PubMed  Article  CAS  Google Scholar 

  28. Nakabayashi, M. et al. Structure of the gene encoding laminaripentaose-producing β-1,3-glucanase (LPHase) of Streptomyces matensis DIC-108. J. Ferment. Bioeng. 85, 459–464 (1998).

    CAS  Article  Google Scholar 

  29. Jamois, F. et al. Glucan-like synthetic oligosaccharides: iterative synthesis of linear oligo-beta-(1,3)-glucans and immunostimulatory effects. Glycobiology 15, 393–407 (2005).

    CAS  PubMed  Article  Google Scholar 

  30. Miyanishi, N., Iwamoto, Y., Watanabe, E. & Odaz, T. Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1. J. Biosci. Bioeng. 95, 192–195 (2003).

    CAS  PubMed  Article  Google Scholar 

  31. Cockburn, D. & Svensson, B. in Carbohydrate Chemistry Vol. 39, 204–221 (The Royal Society of Chemistry, 2013).

  32. Viborg, A. H. et al. A subfamily roadmap for functional glycogenomics of the evolutionarily diverse glycoside hydrolase family 16 (GH16). J. Biol. Chem. 294, 15973–15986 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Juge, N., Payan, F. & Williamson, G. XIP-I, a xylanase inhibitor protein from wheat: a novel protein function. Biochim. Biophys. Acta 1696, 203–211 (2004).

    CAS  PubMed  Article  Google Scholar 

  34. Patil, D. N. et al. Structural investigation of a novel N-acetyl glucosamine binding chi-lectin which reveals evolutionary relationship with class III chitinases. PLoS ONE 8, e63779 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Gerlt, J. A. et al. Enzyme function initiative-enzyme similarity tool (EFI-EST): a web tool for generating protein sequence similarity networks. Biochim. Biophys. Acta 1854, 1019–1037 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Finn, R. D., Clements, J. & Eddy, S. R. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39, W29–W37 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 3150–3152 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Nandi, L. G., Guerra, J. P., Bellettini, I. C., Machado, V. G. & Minatti, E. Properties of aqueous solutions of lentinan in the absence and presence of zwitterionic surfactants. Carbohydr. Polym. 98, 1–7 (2013).

    CAS  PubMed  Article  Google Scholar 

  41. Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428 (1959).

    CAS  Article  Google Scholar 

  42. Dos Santos, C. R. et al. The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans. Biotechnol. Biofuels 11, 223 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  43. Dauter, Z., Dauter, M. & Rajashankar, K. R. Novel approach to phasing proteins: derivatization by short cryo-soaking with halides. Acta Crystallogr. D. 56, 232–237 (2000).

    CAS  PubMed  Article  Google Scholar 

  44. Kabsch, W. XDS. Acta Crystallogr. D. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A. 64, 112–122 (2008).

    CAS  PubMed  Article  Google Scholar 

  46. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D. 58, 1948–1954 (2002).

    PubMed  Article  CAS  Google Scholar 

  47. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D. 53, 240–255 (1997).

    CAS  PubMed  Article  Google Scholar 

  50. Agirre, J. et al. Privateer: software for the conformational validation of carbohydrate structures. Nat. Struct. Mol. Biol. 22, 833–834 (2015).

    CAS  PubMed  Article  Google Scholar 

  51. Franke, D. et al. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212–1225 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Lee, S. et al. CHARMM36 united atom chain model for lipids and surfactants. J. Phys. Chem. B. 118, 547–556 (2014).

    CAS  PubMed  Article  Google Scholar 

  54. Jorgensen, W., Chandrasekhar, J., Madura, J., Impey, R. & Klein, M. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    CAS  Article  Google Scholar 

  55. Martinez, L., Andrade, R., Birgin, E. G. & Martinez, J. M. PACKMOL: a package for building initial configurations for molecular dynamics simulations. J. Comput. Chem. 30, 2157–2164 (2009).

    CAS  PubMed  Article  Google Scholar 

  56. Anandakrishnan, R., Aguilar, B. & Onufriev, A. V. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res. 40, W537–W541 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Darden, T., York, D. & Pedersen, L. Particle mesh Ewald: an Nlog(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    CAS  Article  Google Scholar 

  58. Feller, S. E., Zhang, Y., Pastor, R. W. & Brooks, B. R. Constant pressure molecular dynamics simulation: the Langevin piston method. J. Chem. Phys. 103, 4613–4621 (1995).

    CAS  Article  Google Scholar 

  59. Hoover, W. G. Constant-pressure equations of motion. Phys. Rev. A. 34, 2499–2500 (1986).

    CAS  Article  Google Scholar 

  60. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M.L. Sforça and J.A. Aricetti for the support in data collection of 1H-NMR spectra and lentinan purification, respectively. We thank C.S. Farah for the access and support in the operation of the rotating anode X-ray generator available at the Chemistry Institute, University of São Paulo. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The Stanford Synchrotron Radiation Lights Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS or NIH. We acknowledge the LNLS for the provision of time on the MX2, SAXS1 and SAXS2 beamlines, the Brazilian Biosciences National Laboratory (LNBio) for accessibility to crystallization (Robolab), NMR and spectroscopy facilities and the Brazilian Biorenewables National Laboratory (LNBR) for the use of the characterization of macromolecules facility. LNLS, LNBio and LNBR are operated by the Brazilian Center for Research in Energy and Materials for the Brazilian Ministry for Science, Technology, Innovations and Communications. This research was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant nos. 2015/26982-0 to M.T.M. and 2013/08293-7 to M.S.S.) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant no. 306135/2016-7 to M.T.M.).

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Authors

Contributions

M.T.M. initiated the study and directed the project. S.E.T.G., E.T.P. and M.S.S. devised and performed the molecular dynamic simulations. P.A.C.R.C. and G.F.P. performed the bioinformatics analyses. R.A.S.P. and F.C.G. carried out the mass spectrometry analyses. C.R.S., P.A.C.R.C., E.A.L., F.M., P.S.V., T.L.R.C., L.C., R.L.C., M.P.M., M.N.D., B.P.S. and A.T.J. expressed and purified the enzymes and performed the structural and functional characterization. C.R.S., P.S.V., G.F.P., M.S.S. and M.T.M. wrote the paper with input from P.A.C.R.C. and T.L.R.C. All authors analyzed the results and approved the final version of the manuscript.

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Correspondence to Mario T. Murakami.

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Extended data

Extended Data Fig. 1 Domain organization in the GH128 family.

Domain architecture according to PFAM annotation. Subgroups in which the modular organization is present are indicated in roman numbers. Glyco_hydro_cc (PF11790), Sig-70 (PF04542), CBM6 (PF03422), F5_F8 (PF00754), WSC (PF01822), CBM4 (PF02018), Lectin (PF00652), DUFF (PF18099), Glyco_hydro_16 (PF00722) and PDK (PF00801).

Extended Data Fig. 2 Surface-binding sites in the subgroups IV and VI.

Comparison of the surface-binding sites in the subgroups IV (a and b) and VI (c and d). Relative position of the surface-binding sites to the nearest substrate-binding site (a and c). Protein-carbohydrate interactions (b and d).

Supplementary information

Supplementary Information

Supplementary Tables 1–11 and Figs. 1–30.

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Santos, C.R., Costa, P.A.C.R., Vieira, P.S. et al. Structural insights into β-1,3-glucan cleavage by a glycoside hydrolase family. Nat Chem Biol 16, 920–929 (2020). https://doi.org/10.1038/s41589-020-0554-5

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