Structural characterization of class A β-lactamase CTX-M-15 and class C β-lactamase CMY-2 and their complexes with β-lactam compounds

Parvaneh Ahmadvand

doi:10.7273/000005156

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamases (ESBLs) are Ambler class A cephalosporinases that are commonly associated with Gram-negative, hospital-acquired infections worldwide. The cephamycinase CMY-2 enzyme is an Ambler class C cephalosporinase that is also associated with Gram-negative bacteria. To understand the mechanism of CTX-M-15 and CMY-2 activity, we have determined their crystal structures in complex with several cephalosporin and penicillin compounds, and with a β-lactamase inhibitor, clavulanic acid. The CTX-M-15’s crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of these compounds. The CMY2’s acyl-enzyme crystal structure suggested that residues Ser64, Gln120, Tyr150, Asn152, Glu272, Ala292, Lys315, Thr316, Ser318, Gly320, and Asn346 contribute to both binding and hydrolysis. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant CTX-M-15 structures, we determined that this enzyme has a similar affinity for all beta-lactam compounds [ceftiofur, nitrocefin, desfuroylceftiofur (DFC), and ampicillin], but with lower affinity for clavulanic acid. I propose a catalytic mechanism for hydrolysis of the tested β-lactams and a two-step mechanism for inhibition by clavulanic acid. 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. To study of CMY-2, molecular docking models were used to assess the theoretical interactions of these residues with ampicillin, nitrocefin, ceftiofur, and the ceftiofur metabolites DFC, DFC-dimer, and DFC-cysteine. Thermodynamic and steady-state kinetics data were collected for the wild-type and S64A-mutant enzymes. The helix α-2 of CMY-2 has a conserved catalytic site that includes the nucleophilic Ser64 and the acid/base catalyst Lys67. The β-6 strand (Lys315, Thr316, Ser318) forms an oxyanion hole and loop Ω-8 (Tyr150 and Asn152) that interacts to form the edge of active site. The hydrolysis mechanism of CMY-2 appears to involve the Lys67 as a general base and its conjugate acid that composes the general acid expected for class C β-lactamases. The S64A-mutated enzyme displayed a significant drop in turnover for all tested β-lactams, consistent with Ser64 being a strategic residue in the acylation reaction. Compared to other substrates, the higher magnitude of entropy with ceftiofur is consistent with the greater degree of flexibility or mobility associated with ceftiofur and the binding pocket residues, to which the furfural moiety of ceftiofur contributes.

Structural characterization of class A β-lactamase CTX-M-15 and class C β-lactamase CMY-2 and their complexes with β-lactam compounds

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