Characterization of Interactions between CTX-M-15 and Clavulanic Acid, Desfuroylceftiofur, Ceftiofur, Ampicillin, and Nitrocefin

Abstract

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.

Keywords: CTX-M-15; antibiotic; antimicrobial resistance; crystal structure; inhibition mechanism; inhibitor; β-lactam compounds; β-lactamase.

Figure 1

Chemical structures of five β-lactam compounds. (a) Ceftiofur; (b) Nitrocefin; (c) Desfuroylceftiofur (DFC); (d) Ampicillin; (e) Clavulanic acid. Thiazolidine, β-lactam, aminothiazole, thiophene, penam, and oxazoline rings—and also methoxyimine and furfural groups—are shown with arrows. The figures were generated with the MarvinSketch v.17.28.0. (Budapest, Hungary).

Figure 2

Global structure of CTX-M-15. (a) CTX-M-15 is shown with two domains, α and α/β. The active site is positioned toward the center, showing the catalytic Ser70, Lys73, Ser130, and Glu166. The figure was generated with UCSF Chimera v1.13.1. (San Francisco, CA, USA). (b) The 2D structure of binding pocket of CTX-M-15 contained the residues and water molecules was built by the MarvinSketch v.17.28.0. (Budapest, Hungary).

Figure 3

Active site of CTX-M-15 Clavulanic acid-enzyme complex. (a) Electron density map of CTX-M-15 active site bound to clavulanic acid shown in yellow. 2Fo-Fc map (contoured at 1.0 sigma) is shown in grey which was built by UCSF Chimera v1.13.1 (San Francisco, CA, USA). Residues around the binding pocket of the clavulanic acid-CTX-M-15 crystal structure are depicted as cream, red, blue sticks. (b) 2D structure of clavulanic acid (orange) in the binding pocket of CTX-M-15. All residues have been shown, hydrogen bonds are drawn using dashed lines. The residues that interacted with clavulanic acid are in dark black, residues without interaction are shown in gray. The figure was built by the MarvinSketch v.17.28.0. (Budapest, Hungary).

Figure 4

Active site of CTX-M-15 of the ampicillin-enzyme complex. (a) Electron density map of the CTX-M-15 active site bound to ampicillin in the yellow. 2Fo-Fc map (contoured at 1.0 sigma) is shown in dark gray. Residues around the binding pocket of the ampicillin -CTX-M-15 crystal structure are depicted as cream, red, blue sticks. The figure was generated with UCSF Chimera v1.13.1. (San Francisco, CA, USA). (b) 2D structure of ampicillin (orange) in the binding pocket of CTX-M-15. All residues have been shown, hydrogen bonds are drawn using dashed lines. The residues that interacted with ampicillin are shown in dark black, residues without interaction are depicted in gray. The figure was built by the MarvinSketch v.17.28.0. (Budapest, Hungary).

Figure 5

Active site of CTX-M-15 in DFC-enzyme complex. (a) Electron density map of the CTX-M-15 active site bound to DFC in the yellow color. The 2Fo-Fc map (contoured at 1.0 sigma) is shown in gray. Residues around the binding pocket of the DFC-CTX-M-15 crystal structure are depicted as cream, red, blue sticks. The UCSF Chimera v1.13.1. (San Francisco, CA, USA). built this figure. (b) The 2D structure of DFC (red color) in the binding pocket of CTX-M-15. All residues have been showed, the hydrogen bond drown by dashed lines. The residues had interaction with DFC were in dark black color, and other ones did not have interaction in the gray color. The figure was generated with the MarvinSketch v.17.28.0. (Budapest, Hungary).

Figure 6

Ligand binding pocket of CTX-M-15. (a) Ampicillin is in the binding pocket of enzyme. (b) DFC is in the binding pocket of enzyme. Hydrogen bonds are depicted by block dotted lines and the ligand is in the red color. The figures were generated with the MarvinSketch v.17.28.0. (Budapest, Hungary).

Figure 7

The proposed mechanism for clavulanic acid inhibition of CTX-M-15. Orange colored lines indicate electron transfer and red dashed lines indicate hydrogen bond interactions. Figures were generated using MarvinSketch v.17.28.0. (Budapest, Hungary). (I) Clavulanic acid binding is displayed as a series proton and electron transfers. Lys73 is deprotonated by Glu166 and then the neutral Lys73 activates Ser70 by abstracting the proton from its sidechain. The nucleophilic Ser70 attacks the b-lactam ring. (II) The tetrahedral transition state is stabilized by an oxyanion hole generated with backbones of Ser70 and Ser237. The bridgehead nitrogen atom abstracts a proton from Ser130, and the shared proton is transferred to Ser130 from Lys73, and then to Lys73 from Glu166. (III) The double bond (C6–C9) in the acyl-enzyme intermediate abstracts a proton from a water molecule. The cleavage of the oxazoline ring is followed by abstracting a proton from a water molecule that is protonated by Lys73, and then neutral Lys73 abstracts a proton from the bridgehead nitrogen atom. (IV) The negatively charged Glu166 abstracts a proton from a water molecule, the activated water molecule removes an acidic hydrogen on the a-carbon (C3). (V) trans-imine-enzyme is stabilized by several hydrogen bond interactions.