8 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
Crotonases: Natureâs exceedingly convertible catalysts
YesThe crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, ι,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by ι-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis.Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trus
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
19F NMR Monitoring of Reversible Protein PostâTranslational Modifications: Class D βâLactamase Carbamylation and Inhibition
Bacterial production of βâlactamases with carbapenemase activity is a global health threat. The active sites of classâ
D carbapenemases such as OXAâ48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this postâtranslational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19Fâ
NMR spectroscopy to monitor the carbamylation state of 19Fâlabelled OXAâ48. This method was used to investigate the interactions of OXAâ48 with clinically used serine βâlactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19Fâlabelled OXAâ48 provide a structural rationale for the sensitivity of the 19F label to active site interactions. The overall results demonstrate the use of 19Fâ
NMR to monitor reversible covalent postâtranslational modifications.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