5 research outputs found
Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation
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 Î2 (enamine) and/or (R)-Î1 (imine) products. Rapid non-enzymatic tautomerisation of the Î2 product to the (R)-Î1 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.FWN â Publicaties zonder aanstelling Universiteit Leide
A New Mechanism for βâLactamases: Class D Enzymes Degrade 1βâMethyl Carbapenems through Lactone Formation
βâLactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acylâenzyme intermediate. We show that classâ
D βâlactamases also degrade clinically used 1βâmethylâsubstituted carbapenems through the unprecedented formation of a carbapenemâderived βâlactone. βâLactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acylâenzyme intermediate. The carbapenemâderived lactone products inhibit both serine βâlactamases (particularly classâ
D) and metalloâβâlactamases. These results define a new mechanism for the classâ
D carbapenemases, in which a hydrolytic water molecule is not required.FWN â Publicaties zonder aanstelling Universiteit Leide
A New Mechanism for βâLactamases: Class D Enzymes Degrade 1βâMethyl Carbapenems through Lactone Formation
βâLactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acylâenzyme intermediate. We show that classâ
D βâlactamases also degrade clinically used 1βâmethylâsubstituted carbapenems through the unprecedented formation of a carbapenemâderived βâlactone. βâLactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acylâenzyme intermediate. The carbapenemâderived lactone products inhibit both serine βâlactamases (particularly classâ
D) and metalloâβâlactamases. These results define a new mechanism for the classâ
D carbapenemases, in which a hydrolytic water molecule is not required.FWN â Publicaties zonder aanstelling Universiteit Leide
Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation
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 Î2 (enamine) and/or (R)-Î1 (imine) products. Rapid non-enzymatic tautomerisation of the Î2 product to the (R)-Î1 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
Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation
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 Î2 (enamine) and/or (R)-Î1 (imine) products. Rapid non-enzymatic tautomerisation of the Î2 product to the (R)-Î1 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