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Substrate Specificity of OXA-48 after β5−β6 Loop Replacement
journal contribution
posted on 2020-03-19, 14:08 authored by Laura Dabos, Agustin Zavala, Rémy A. Bonnin, Oliver Beckstein, Pascal Retailleau, Bogdan I. Iorga, Thierry NaasOXA-48
carbapenemase has rapidly spread in many countries worldwide with
several OXA-48-variants being described, differing by a few amino
acid (AA) substitutions or deletions, mostly in the β5−β6
loop. While single AA substitutions have only a minor impact on OXA-48
hydrolytic profiles, others with 4 AA deletions result in loss of
carbapenem hydrolysis and gain of expanded-spectrum cephalosporin
(ESC) hydrolysis. We have replaced the β5−β6 loop
of OXA-48 with that of OXA-18, a clavulanic-acid inhibited oxacillinase
capable of hydrolyzing ESCs but not carbapenems. The hybrid enzyme
OXA-48Loop18 was able to hydrolyze ESCs and carbapenems (although
with a lower kcat), even though the β5−β6
loop was longer and its sequence quite different from that of OXA-48.
The kinetic parameters of OXA-48Loop18 were in agreement with the
MIC values. X-ray crystallography and molecular modeling suggest that
the conformation of the grafted loop allows the binding of bulkier
substrates, unlike that of the native loop, expanding the hydrolytic
profile. This seems to be due not only to differences in AA sequence,
but also to the backbone conformation the loop can adopt. Finally,
our results provide further experimental evidence for the role of
the β5−β6 loop in substrate selectivity of OXA-48-like
enzymes and additional details on the structure–function relationship
of β-lactamases, demonstrating how localized changes in these
proteins can alter or expand their function, highlighting their plasticity.