Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site

Abstract

β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.

Negative Impact of Carbapenem Methylation on the Reactivity of β-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses

“…Second, comparison of carbapenems with different side chains revealed only minor differences in the acylation efficacy (6). The same observation was made for cephems and penams (4), with, in the former case, the noticeable exception of nitrocefin, which acylates Ldt fm more rapidly than imipenem, due to the unusual electron-withdrawing property of its side chain (7). Third, the affinity of a catalytically inactive form of Ldt fm , obtained by replacing the catalytic Cys with Ala, revealed no significant difference in the K D (equilibrium dissociation constant) values for representatives of the carbapenems (ertapenem), cephems (ceftriaxone), and penams (ampicillin), i.e., 50, 44, and 79 mM, respectively (4).…”

“…Optimization of ␤-lactams for inactivation of L,Dtranspeptidases, which have recently found potential applications in the treatment of infections due to mycobacteria (23)(24)(25), should therefore focus on improving reactivity while preserving sufficient drug stability, although toxicity issues may arise from acylation of human enzymes. Additional properties, including limited acyl enzyme hydrolysis (4) and the irreversibility of the ␤-lactam ring opening (7), are also important for antibacterial activity.…”

Preparation and Evaluation of the ZnO NP–Ampicillin/Sulbactam Nanoantibiotic: Optimization of Formulation Variables Using RSM Coupled GA Method and Antibacterial Activities

“…ZnO NP hampers the ESBL enzyme present in the cytoplasm and also changes the permeability of the cell wall of bacteria. This in turn might enhance the entry of Ams into K. pneumoniae cells, and allow for the antibiotic to perform its own mechanism of action i.e., inhibition of cell wall synthesis by irreversibly binding to the active site of transpeptidase enzyme [57]. Hence, both ZnO NP and Ams works synergistically against the Ams-resistant K. pneumoniae.…”

Выбор Антибактериального Препарата При Инфекциях Мочевыделительной Системы У Детей

“…Період напіввиведення: найбільш тривалий період напіввиведення (3,7 години) порівняно з іншими пероральними цефалоспоринами. При тяжкій нирковій недостатності (кліренс креатиніну від 5 до 20 мл/хв) період напіввиведення збільшується в середньому до 11,5 години, що вимагає корекції дози [7].…”