J. Ross, M. J. Hoopman, F. Kullmer, O. Al-Jourani, A. Silale, M. M. Osman, Z. Chen, H. R. Bridges, K. J. Garnham, C. Morland, A. Layton, J.-L. Reyre, J. Turkenburg, S. Hart, A. Solovyova, A. Porter, A. Basle, J. D. C. Codee, S. J. Williams, P. J. Moynihan, H. S. Overkleeft, J. N. Blaza, E. C. Lowe
Mycobacteria synthesise the unusual glycan D-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 D-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo-alpha-D-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 alpha-D-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 alpha-D-arabinofuranosidase activity in complex biological samples.