Cefotaximase-Munich (CTX-M) extended-spectrum
beta-lactamases (ESBLs) are commonly associated with Gram-negative, hospital-acquired
infections worldwide. Several
beta-lactamase inhibitors, such as
clavulanate, are used to inhibit the activity of these
enzymes. To understand the mechanism of CTX-M-15 activity, we have determined the crystal structures of CTX-M-15 in complex with two specific classes of
beta-lactam compounds,
desfuroylceftiofur (DFC) and
ampicillin, and an inhibitor,
clavulanic acid. The crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of those compounds. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant structures, we determined that CTX-M-15 has a similar affinity for all
beta-lactam compounds (
ceftiofur,
nitrocefin, DFC, and
ampicillin), but with lower affinity for
clavulanic acid. A catalytic mechanism for tested β-
lactams and two-step inhibition mechanism of
clavulanic acid were proposed. CTX-M-15 showed a higher activity toward DFC and
nitrocefin, but significantly lower activity toward
ampicillin and
ceftiofur. The interaction between CTX-M-15 and both
ampicillin and
ceftiofur displayed a higher entropic but lower enthalpic effect, compared with DFC and
nitrocefin. DFC, a metabolite of
ceftiofur, displayed lower entropy and higher enthalpy than
ceftiofur. This finding suggests that compounds containing
amine moiety (e.g.,
ampicillin) and the
furfural moiety (e.g.,
ceftiofur) could hinder the hydrolytic activity of CTX-M-15.