Abstract
β-Lactamases are a major threat to the clinical use of carbapenems, which are often antibiotics of last resort. Despite this, the reaction outcomes and mechanisms by which β-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a β-lactam ring fused to a pyrroline ring. Following β-lactamase-mediated opening of the β-lactam, the pyrroline may interconvert between an enamine (2-pyrroline) form and two epimeric imine (1-pyrroline) forms; previous crystallographic and spectroscopic studies have reported all three of these forms in the contexts of hydrolysis by different β-lactamases. As we show by NMR spectroscopy, the serine β-lactamases (KPC-2, SFC-1, CMY-10, OXA-23, and OXA-48) and metallo-β-lactamases (NDM-1, VIM-1, BcII, CphA, and L1) tested all degrade carbapenems to preferentially give the Δ2 (enamine) and/or (R)-Δ1 (imine) products. Rapid non-enzymatic tautomerisation of the Δ2 product to the (R)-Δ1 product prevents assignment of the nascent enzymatic product by NMR. The observed stereoselectivity implies that carbapenemases control the form of their pyrroline ring intermediate(s)/product(s), thereby preventing pyrroline tautomerisation from inhibiting catalysis.
Highlights
IntroductionIn the products derived from carbapenem degradation (i.e., hydrolysis and β-lactone products; Fig. 1), the carbapenem pyrroline ring exists in an equilibrium between an enamine (2-pyrroline) form (Δ2) and two www.nature.com/scientificreports/
As we show by NMR spectroscopy, the serine β-lactamases (KPC-2, Serratia fonticola carbapenemase-1 (SFC-1), CMY-10, OXA-23, and OXA-48) and metallo-β-lactamases (NDM-1, VIM1, BcII, carbapenemase hydrolysing Aeromonas (CphA), and L1) tested all degrade carbapenems to preferentially give the Δ2 and/or (R)-Δ1 products
Unclear whether these structures represent catalytically viable complexes which are susceptible to hydrolysis, or if the observed form of the pyrroline ring reflects slow/inhibited degradation of the acyl-enzyme complex(es)
Summary
In the products derived from carbapenem degradation (i.e., hydrolysis and β-lactone products; Fig. 1), the carbapenem pyrroline ring exists in an equilibrium between an enamine (2-pyrroline) form (Δ2) and two www.nature.com/scientificreports/. Epimeric imine (1-pyrroline) forms [(R)-Δ1 and (S)-Δ1; Fig. 1]2,5 This equilibrium has potential to extend to the acyl-enzyme complexes derived from carbapenems and SBLs, where all three forms of the pyrroline ring have been reported in crystallographic analyses with different enzymes[7,8,9,10,11]. Interconversion between the different pyrroline forms appears to be time-dependent, and may occur slowly in the enzyme active site; crystallographic studies of the acyl-enzyme complex derived from the SBL BlaC with the carbapenem ertapenem suggest that the pyrroline ring is initially present in the Δ2 enamine form, and tautomerises to the (S)-Δ1 imine form over time[7]
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