Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures

Ross, J.; Hoopman, M. J.; Küllmer, F.; Al-Jourani, O.; Silale, A.; Osman, M. M.; Chen, Z.; Bridges, H. R.; Garnham, K. J.; Morland, C.; Reyre, J.-L.; Layton, A. J.; Turkenburg, J.; Hart, S.; Solovyova, A.; Porter, A.; Basle, A.; Codee, J. D. C.; Williams, S. J.; Moynihan, P. J.; Overkleeft, H. S.; Blaza, J. N.; Lowe, E. C.

doi:10.64898/2026.08.13.744632

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bioRxiv · 10.64898/2026.08.13.744632

Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures

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Abstract

Mycobacteria synthesise the unusual glycan [x1D05]-arabinan as a major component of the cell wall glycoconjugates arabinogalactan (AG) and lipoarabinomannan (LAM). We previously identified Dysgonomonas gadei, a member of the Bacteroidota, as capable of complete [x1D05]-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo--[x1D05]-arabinofuranosidase activity against AG and LAM, although their linkage specificities were unknown. Here, using defined synthetic substrates, we show that the three enzymes possess distinct linkage preferences. We also develop -[x1D05]-arabinofuranosyl cyclophellitol aziridines as covalent inhibitors and activity-based probes for GH172 enzymes. X-ray crystallography and cryo-EM to reveal strikingly different quaternary assemblies across the three homologues, while a 1.5 [A] cryo-EM structure of dodecameric Dg67 covalently modified by an aziridine inhibitor identifies the catalytic nucleophile and provides direct structural support for a retaining mechanism. A BODIPY-tagged aziridine probe selectively labelled the three GH172 enzymes in D. gadei cell lysates. Together, these findings define functional and structural diversity within GH172 and establish chemical probes for profiling -[x1D05]-arabinofuranosidase activity in complex biological samples.

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Ross, J., Hoopman, M. J., Küllmer, F., Al-Jourani, O., Silale, A., Osman, M. M., Chen, Z., Bridges, H. R., Garnham, K. J., Morland, C., Reyre, J.-L., Layton, A. J., Turkenburg, J., Hart, S., Solovyova, A., Porter, A., Basle, A., Codee, J. D. C., Williams, S. J., Moynihan, P. J., Overkleeft, H. S., Blaza, J. N., Lowe, E. C.. 2026-08-18. Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures. https://doi.org/10.64898/2026.08.13.744632

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