posted on 2023-12-05, 16:08authored byValentina Borlandelli, Wendy Offen, Olga Moroz, Alba Nin-Hill, Nicholas McGregor, Lars Binkhorst, Akihiro Ishiwata, Zachary Armstrong, Marta Artola, Carme Rovira, Gideon J. Davies, Herman S. Overkleeft
GH127 and GH146 microorganismal
retaining β-l-arabinofuranosidases,
expressed by human gut microbiomes, feature an atypical catalytic
domain and an unusual mechanism of action. We recently reported that
both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by β-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated
cysteine as a catalytic nucleophile, where in most retaining GH families,
an aspartate or glutamate is employed. This work presents a panel
of β-l-arabinofuranosyl cyclophellitol
epoxides and aziridines as mechanism-based BtGH146/HypBA1
inhibitors and activity-based probes. The β-l-arabinofuranosyl cyclophellitol aziridines both inhibit
and label β-l-arabinofuranosidase efficiently (however
with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray
structural analysis of the unmodified β-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes,
which were shown to react by nucleophilic opening of the aziridine,
at the pseudoanomeric carbon, by the active site cysteine nucleophile
to form a stable thioether bond. Altogether, our activity-based probes
may serve as chemical tools for the detection and identification of
low-abundance β-l-arabinofuranosidases in complex biological
samples.