Expansion of the diversity of dispersin scaffolds

Alexandra Males; Olga V. Moroz; Elena Blagova; Astrid Munch; Gustav H. Hansen b; Annette H. Johansen; Lars H. Østergaard; Dorotea R. Segura; Alexander Eddenden; Anne V. Due; Martin Gudmand; Jesper Salomon; Sebastian R. Sørensen; João Paulo L. Franco Cairo; Mark Nitz; Roland A. Pache; Rebecca M. Vejborg; Gideon J. Davies; Keith S. Wilson

doi:10.1107/S205979832500110X

Expansion of the diversity of dispersin scaffolds

Alexandra Males, Olga V. Moroz, Elena Blagova, Astrid Munch, Gustav H. Hansen b, Annette H. Johansen, Lars H. Østergaard, Dorotea R. Segura, Alexander Eddenden, Anne V. Due, Martin Gudmand, Jesper Salomon, Sebastian R. Sørensen, João Paulo L. Franco Cairo, Mark Nitz, Roland A. Pache, Rebecca M. Vejborg, Gideon J. Davies, Keith S. Wilson

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-d-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.

Original language	English
Pages (from-to)	130-146
Number of pages	17
Journal	Acta Crystallographica Section D: Structural Biology
Volume	81
Issue number	3
DOIs	https://doi.org/10.1107/S205979832500110X https://doi.org/10.1107/S205979832500110X
Publication status	Published - 28 Feb 2025

Bibliographical note

Publisher Copyright:
© Alexandra Males et al. 2025.

Keywords

enzyme catalysis
GH20
glycoside hydrolases
poly-N-acetylglucosamine
protein crystallography

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10.1107/S205979832500110X

Expansion of the diversity of dispersin scaffoldsFinal published version, 13.8 MBLicence: CC BY

Cite this

Males, A., Moroz, O. V., Blagova, E., Munch, A., b, G. H. H., Johansen, A. H., Østergaard, L. H., Segura, D. R., Eddenden, A., Due, A. V., Gudmand, M., Salomon, J., Sørensen, S. R., Cairo, J. P. L. F., Nitz, M., Pache, R. A., Vejborg, R. M., Davies, G. J., & Wilson, K. S. (2025). Expansion of the diversity of dispersin scaffolds. Acta Crystallographica Section D: Structural Biology, 81(3), 130-146. https://doi.org/10.1107/S205979832500110X, https://doi.org/10.1107/S205979832500110X

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title = "Expansion of the diversity of dispersin scaffolds",

abstract = "Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-d-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.",

keywords = "enzyme catalysis, GH20, glycoside hydrolases, poly-N-acetylglucosamine, protein crystallography",

author = "Alexandra Males and Moroz, {Olga V.} and Elena Blagova and Astrid Munch and b, {Gustav H. Hansen} and Johansen, {Annette H.} and {\O}stergaard, {Lars H.} and Segura, {Dorotea R.} and Alexander Eddenden and Due, {Anne V.} and Martin Gudmand and Jesper Salomon and S{\o}rensen, {Sebastian R.} and Cairo, {Jo{\~a}o Paulo L. Franco} and Mark Nitz and Pache, {Roland A.} and Vejborg, {Rebecca M.} and Davies, {Gideon J.} and Wilson, {Keith S.}",

note = "Publisher Copyright: {\textcopyright} Alexandra Males et al. 2025.",

year = "2025",

month = feb,

day = "28",

doi = "10.1107/S205979832500110X",

language = "English",

volume = "81",

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journal = "Acta Crystallographica Section D: Structural Biology",

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Males, A , Moroz, OV , Blagova, E, Munch, A, b, GHH, Johansen, AH, Østergaard, LH, Segura, DR, Eddenden, A, Due, AV, Gudmand, M, Salomon, J, Sørensen, SR, Cairo, JPLF, Nitz, M, Pache, RA, Vejborg, RM, Davies, GJ & Wilson, KS 2025, 'Expansion of the diversity of dispersin scaffolds', Acta Crystallographica Section D: Structural Biology, vol. 81, no. 3, pp. 130-146. https://doi.org/10.1107/S205979832500110X, https://doi.org/10.1107/S205979832500110X

TY - JOUR

T1 - Expansion of the diversity of dispersin scaffolds

AU - Males, Alexandra

AU - Moroz, Olga V.

AU - Blagova, Elena

AU - Munch, Astrid

AU - b, Gustav H. Hansen

AU - Johansen, Annette H.

AU - Østergaard, Lars H.

AU - Segura, Dorotea R.

AU - Eddenden, Alexander

AU - Due, Anne V.

AU - Gudmand, Martin

AU - Salomon, Jesper

AU - Sørensen, Sebastian R.

AU - Cairo, João Paulo L. Franco

AU - Nitz, Mark

AU - Pache, Roland A.

AU - Vejborg, Rebecca M.

AU - Davies, Gideon J.

AU - Wilson, Keith S.

PY - 2025/2/28

Y1 - 2025/2/28

N2 - Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-d-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.

AB - Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-d-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.

KW - enzyme catalysis

KW - GH20

KW - glycoside hydrolases

KW - poly-N-acetylglucosamine

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DO - 10.1107/S205979832500110X

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JO - Acta Crystallographica Section D: Structural Biology

JF - Acta Crystallographica Section D: Structural Biology

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ER -

Expansion of the diversity of dispersin scaffolds

Abstract

Bibliographical note

Keywords

Access to Document

Other files and links

RS Research Professorship

X-ray Diffraction Equipment for Macromolecular Crystallography at York

Cite this

Expansion of the diversity of dispersin scaffolds

Abstract

Bibliographical note

Keywords

Access to Document

Other files and links

Projects

RS Research Professorship

X-ray Diffraction Equipment for Macromolecular Crystallography at York

Cite this