The rise of multi-
and even totally antibiotic resistant forms
of Mycobacterium tuberculosis underlines the need
for new antibiotics. The pathogen is resistant to β-lactam compounds
due to its native serine β-lactamase, BlaC. This resistance
can be circumvented by administration of a β-lactamase inhibitor.
We studied the interaction between BlaC and the inhibitor clavulanic
acid. Our data show hydrolysis of clavulanic acid and recovery of
BlaC activity upon prolonged incubation. The rate of clavulanic acid
hydrolysis is much higher in the presence of phosphate ions. A specific
binding site for phosphate is identified in the active site pocket,
both in the crystalline state and in solution. NMR spectroscopy experiments
show that phosphate binds to this site with a dissociation constant
of 30 mM in the free enzyme. We conclude that inhibition of BlaC by
clavulanic acid is reversible and that phosphate ions can promote
the hydrolysis of the inhibitor.
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