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 Ldt_fm 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 Ldt_fm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.

Figure 1

Reactions catalyzed by the L,D-transpeptidase Ldt_fm with nitrocefin, imipenem, and ceftriaxone. The figure also shows the structures of cephalothin and ampicillin. The base (B) that provides a proton to the β-lactam nitrogen of imipenem has not been identified^, .

Figure 2

Determination by spectrophotometry of the acylation of Ldt_fm by nitrocefin. (A) Absorbance spectra of various forms of nitrocefin (50 µM). Blue, native nitrocefin; orange, nitrocefin hydrolyzed by BlaC; grey, nitrocefin in the acyl-enzyme formed with Ldt_fm. (B) Titration of the active site of Ldt_fm (22 µM) by nitrocefin. (C and D) Mass spectra of Ldt_fm and of the acyl-enzyme formed with nitrocefin. The average mass of Ldt_fm (E) and of the acyl-enzyme (EN*) were deduced from the m/z ratios of the [M + 17 H]¹⁷⁺ and [M + 18 H]¹⁸⁺ ions. The calculated mass of nitrocefin, Ldt_fm, and of the acyl-enzyme were 516.5 Da, 16,639.3 Da, and 17,155.8 Da, respectively.

Figure 3

Kinetics of Ldt_fm acylation by nitrocefin and of hydrolysis of the resulting acyl-enzyme. (A) Stopped-flow kinetics of acylation of Ldt_fm (11 µM) by nitrocefin (25, 50, 75, and 100 µM) in 100 mM sodium phosphate buffer (pH 6.0) at 20 °C. The value of k _obs was determined by fitting Equation 1 to data. E₀, concentration of native Ldt_fm at t = 0. (B) Variation of k _obs as a function of the concentration of nitrocefin. (C) Hydrolysis of a fixed concentration of nitrocefin (50 µM) by Ldt_fm at various concentrations (0 to 22 µM). (D) The initial rate of hydrolysis (v _i) was plotted as a function of the concentration of Ldt_fm and the value of k ₃ was determined by linear regression (Equation 2) considering that Ldt_fm is fully acylated under these conditions.

Figure 4

Competitive acylation of Ldt_fm by nitrocefin and imipenem. One syringe of the stopped-flow apparatus contained nitrocefin (100 µM) and imipenem (128 µM) in 100 mM sodium phosphate buffer (pH 6.0). The second syringe contained Ldt_fm (44 µM) in the same buffer. Equal volumes from syringes 1 and 2 were injected into the cuvette of the spectrophotometer and the absorbance was recorded at 486 nm. Concentrations indicated in the Figure take into account the two-fold dilution. Panels A and B represent the same kinetics for two timescales (0–6 s and 0–5000 s, respectively). In panel a, the increase in the absorbance at 486 nm mainly results from the rupture of the β-lactam ring of nitrocefin upon acylation of Ldt_fm. In panel B, further increase in the absorbance observed in the absence of imipenem (upper curve) results from slow hydrolysis of nitrocefin. In the presence of imipenem, recyclization of the β-lactam ring of nitrocefin accounts for the decrease in the absorbance.

Figure 5

Detection of nitrocefin recyclization based on competitive acylation of unlabeled and labeled Ldt_fm. (A) Schematic representation of the reactions. Resealing of the β-lactam ring of nitrocefin regenerates the native form of the drug (Re-cyclized nitrocefin) that leaves the Ldt_fm active site and competitively acylates Ldt_fm or¹³C-¹⁵N-labeled Ldt_fm. (B) Uniformly¹³C- and¹⁵N-labeled Ldt_fm (2.2 nmole) was incubated with nitrocefin (1.5 nmole) for 1 min at 20 °C in 100 µl of 5 mM sodium phosphate buffer pH 6.0. The spectrum of the protein revealed ca. 82% acylation (time = 0). Unlabeled Ldt_fm (2.2 nmole in 1.46 µl) was added and mass spectra were recorded at 300 s and 600 s. The relative abundance of the acylated forms of Ldt_fm was deduced from the relative intensity of the peaks.