Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures
Year: 2026
Type: preprint
Abstract: Mycobacteria synthesise the unusual glycan ᴅ-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 ᴅ-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo-α-ᴅ-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 α-ᴅ-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 Å 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 α-ᴅ-arabinofuranosidase activity in complex biological samples.
Authors: Jennifer Ross, Moescha J. Hoopman, Florian Küllmer, Omar Al-Jourani, Augustinas Silale, Morwan M. Osman, Zongjia Chen, Hannah R. Bridges, Kaspar J. Garnham, Carl Morland, Jean-Lou Reyre, Abigail J. Layton, Johan Turkenburg, Sam Hart, Alexandra Solovyova, Andrew Porter, Arnaud Baslé, Jeroen D. C. Codée, Spencer J. Williams, Patrick J. Moynihan, Herman S. Overkleeft, James N. Blaza, Elisabeth C. Lowe
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: green
Awards: An imaging filter to enable high-resolution cryogenic electron tomography (cryo-ET) at York, DESIGN OF CONFORMATIONAL AND COVALENT ENDOGLYCOSIDASE INHIBITORS FOR BIOMEDICINE AND BIOTECHNOLOGY, G4841225454, Exploring the structure-function landscape of a multimeric family of glycoside hydrolases, G5363839104, Understanding the role of D-arabinanases in mycobacterial physiology and pathogenicity, G7641710749, Investigating the molecular basis of Heparan Sulfate biosynthesis, G8274737442
OpenAlex ID: W7203528191