Studies on the Reactions of Biapenem with VIM Metallo β-Lactamases and the Serine β-Lactamase KPC-2

Abstract

Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.

Keywords: antimicrobial resistance; biapenem; carbapenems; metallo-β-lactamases; serine-β-lactamases.

Figure 1

Carbapenem hydrolysis by β-lactamases. (A) It is proposed that SBLs and MBLs hydrolyse carbapenems to produce an enamine (Δ²-pyrroline), which during efficient catalysis in solution isomerises to give (2R)- and (2S)-Δ¹-imine products. Both enamine and (2S)- and (2R)-imine carbapenem-derived ligands have been observed by crystallography at MBL/SBL/transpeptidase active sites. Lactones can also be formed, at least, in the case of class D SBLs. (B) Classes of clinically used β-lactam antibiotics (for carbapenems R¹ = H or Me). (C) Examples of clinically used carbapenems; note the unusual positively charged bicyclic bicyclotriazolium C2 side chain of biapenem.

Figure 2

High-field regions of ¹H NMR spectra (600 MHz) for reaction of biapenem with the MBLs VIM-1 or VIM-2, or the SBL KPC-2. Biapenem (5 mM, green) was treated with the purified β-lactamase (280 nM, 30 min) in 50 mM of sodium phosphate at pH 7.6 (with 10% v/v D₂O). Formation of enamine (pink) and (2R)-imine and (2S)-imine (purple and blue, respectively) products was observed in all three cases. Time-course analyses imply the (2S)-imine is the major and ‘thermodynamic’ product (Figures S1–S4). The slowest rate of biapenem turnover was observed with KPC-2 (Figure S1), with increasing rates observed for VIM-1 (Figure S2), and VIM-2 (Figure S3).

Figure 3

Electron density maps for biapenem-derived products in complex with VIM-2. (2S)-Imine: pale green balls and sticks; enamine: pink balls and sticks. (A,C) show an OMIT map (green mesh) of mFo-DFc contoured to 3σ (PDB: 6Y6J). (B,D) show the 2mFo-DFc difference map in grey mesh, contoured to 1σ. Note the weak density for the biapenem-derived bicyclotriazolium sidechain, indicating the presence of multiple conformations.

Figure 4

Views of a crystal structure of VIM-2 complexed with biapenem-derived ligands (PDB 6Y6J). (A) Interactions occurring between the (2S)-imine (green) and enamine (pink) products at the VIM-2 active site. Note that both products interact via their C3 carboxylate with Arg228 and that the sulfur of the thioether sidechain of the enamine is positioned adjacent to the primary amide of Asn233. (B) The (2S)-imine and enamine tautomers were each modelled at 50% occupancy.