Avibactam
is a potent diazobicyclooctane inhibitor of class A and
C β-lactamases. The inhibitor also exhibits variable activity
against some class D enzymes from Gram-negative bacteria; however,
its interaction with recently discovered class D β-lactamases
from Gram-positive bacteria has not been studied. Here, we describe
microbiological, kinetic, and mass spectrometry studies of the interaction
of avibactam with CDD-1, a class D β-lactamase from the clinically
important pathogen Clostridioides difficile, and
show that avibactam is a potent irreversible mechanism-based inhibitor
of the enzyme. X-ray crystallographic studies at three time-points
demonstrate the rapid formation of a stable CDD-1-avibactam acyl–enzyme
complex and highlight differences in the anchoring of the inhibitor
by class D enzymes from Gram-positive and Gram-negative bacteria.
Class D β-lactamases have risen
to notoriety due to their
wide spread in bacterial pathogens, propensity to inactivate clinically
important β-lactam antibiotics, and ability to withstand inhibition
by the majority of classical β-lactamase inhibitors. Understanding
the catalytic mechanism of these enzymes is thus vitally important
for the development of novel antibiotics and inhibitors active against
infections caused by antibiotic-resistant bacteria. Here we report
an in crystallo time-resolved study of the interaction
of the class D β-lactamase CDD-1 from Clostridioides
difficile with the diazobicyclooctane inhibitor, avibactam.
We show that the catalytic carboxylated lysine, a residue that is
essential for both acylation and deacylation of β-lactams, is
sequestered within an internal sealed pocket of the enzyme. Time-resolved
snapshots generated in this study allowed us to observe decarboxylation
of the lysine and movement of CO2 and water molecules through
a transient channel formed between the lysine pocket and the substrate
binding site facilitated by rotation of the side chain of a conserved
leucine residue. These studies provide novel insights on avibactam
binding to CDD-1 and into the catalytic mechanism of class D β-lactamases
in general.
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