RESUMO
ß-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 ß-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a ß-lactam ring fused to a pyrroline ring. Following ß-lactamase-mediated opening of the ß-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 ß-lactamases. As we show by NMR spectroscopy, the serine ß-lactamases (KPC-2, SFC-1, CMY-10, OXA-23, and OXA-48) and metallo-ß-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.
