Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures
TL;DR
These findings define functional and structural diversity within GH172 and establish chemical probes for profiling α-ᴅ-arabinofuranosidase activity in complex biological samples.
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.