Reversible Inactivation of a Peptidoglycan Transpeptidase by A

www.nature.com/scientificreports OPEN Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by Received: 11 May 2017 Accepted: 17 July 2017 β-lactam-ring recyclization in the Published: xx xx xxxx enzyme active site Zainab Edoo, Michel Arthur & Jean-Emmanuel Hugonnet 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 nitrocefn 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 nitrocefn. Ring strain is therefore not sufcient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics. β-lactams remain the corner stone of antibacterial chemotherapy 76 years afer the frst therapeutic use of an antibiotic, namely penicillin, in 1941. β-lactams kill bacteria by inactivating the D,D-transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis1. Te latter cell wall polymer is a giant macromolecule of 109 to 1010 daltons that completely surrounds bacterial cells and plays an essential role in counteracting the osmotic pressure of the cytoplasm during the entire cell cycle including cell division2. β-lactams are structure analogues of the peptidoglycan precursors and act as suicide substrates of the transpeptidases3. Te enzymes catalyze formation of peptidoglycan cross-links in a two-step reaction4. In the frst step, the D,D-transpeptidases interact with an acyl donor containing a pentapeptide stem ending in D-Ala4-D-Ala5, leading to the release of D-Ala5 and to the formation of an ester bond between the carbonyl of D-Ala4 and the catalytic Ser residue of the enzymes. In the second step, nucleophilic attack of the resulting acyl-enzyme by the acyl acceptor generates the peptidoglycan cross-link. Similar to the frst step of the transpeptidation reaction, nucleophilic attack of the carbon carbonyl of β-lactams by the catalytic Ser leads to rupture of the amide bond of the β-lactam ring and inactivation of the D,D-transpeptidases. Te resulting acyl-enzymes are highly stable, with typical half-lives in the order of several hours, due to low hydrolysis rates. For this reason, the inactivation reaction is considered as irreversible since recovery of enzyme activity through hydrolysis of the drug-enzyme adduct occurs at a timescale that is too large to compromise antibacterial activity. Te D,D-transpeptidases have been historically referred to as penicillin-binding proteins (PBPs) since these enzymes were routinely identifed by covalent labeling with radioactive penicillin followed by gel electropho- resis1. PBPs belong to a super family of mechanistically and structurally related active-site serine enzymes that also comprise D,D-carboxypeptidases, endopeptidases, and most β-lactamases responsible for β-lactam resist- ance by drug detoxifcation3, 5. Te acyl-enzymes formed by the acylation of the Ser residue of β-lactamases are INSERM UMRS 1138, Sorbonne Universités, UPMC Univ Paris 06; Sorbonne Paris Cité, Université Paris Descartes, Université Paris Diderot; Centre de Recherche des Cordeliers, 75006, Paris, France. Correspondence and requests for materials should be addressed to M.A. (email: [email protected]) or J.-E.H. (email: Jean-emmanuel. [email protected]) SCIENTIFIC REPORTS | 7: 9136 | DOI:10.1038/s41598-017-09341-8 1 www.nature.com/scientificreports/ Figure 1. Reactions catalyzed by the L,D-transpeptidase Ldtfm with nitrocefn, imipenem, and cefriaxone. Te fgure also shows the structures of cephalothin and ampicillin. Te base (B) that provides a proton to the β-lactam nitrogen of imipenem has not been identifed23, 26. rapidly hydrolyzed leading to turnover and drug detoxifcation. More recently, the family of β-lactam-interacting enzymes has been enriched by the detection of active-site cysteine L,D-transpeptidases that by-pass the D,D-transpeptidase activity of PBPs in β-lactam-resistant mutants of Enterococcus faecium and Escherichia coli6, 7. Tese L,D-transpeptidases were also found to be the main peptidoglycan cross-linking enzymes in Clostridium difcile and in mycobacteria8. Te L,D-transpeptidases are structurally unrelated to active-site Ser PBPs and cleave the L-Lys3-D-Ala4 or diaminopymelate3-D-Ala4 peptide bond of an acyl donor containing a tetrapeptide stem9, 10. In spite of this diference in substrate specifcity, L,D-transpeptidases interact with β-lactams belonging to the carbapenem class11 (Fig. 1) that are currently being evaluated for the treatment of tuberculosis12 and of pulmonary infections due to Mycobacterium abscessus in cystic fbrosis patients13. Te L,D-transpeptidases are also acylated by β-lactams belonging to the cephalosporin and penam classes but this does not lead to target inactivation and antibacterial activity since the corresponding adducts are prone to hydrolysis14. Due to the strain of the four-membered ring of β-lactams, the acylation reaction is considered as irreversible in the sense that re-sealing of the scissile amide bond of the β-lactam ring is not thought to occur at any signifcant rate15, 16. In this study, we have critically evaluated this postulate by studying the acylation of the L,D-transpeptidase Ldtfm from E. faecium by nitrocefn, a chromogenic cephalosporin. We show that this drug-enzyme combination displays high acylation and low hydrolysis rates leading to acyl-enzyme accumulation. Tis ofered the possibility to assay for recyclization of the β-lactam ring by two approaches based on the use of a competing β-lactam or a competing enzyme. We provide evidence that recyclization of the β-lactam ring of nitrocefn in the acyl-enzyme regenerates the native drug that can freely difuse out of the Ldtfm active site. Tese data indicate that the irreversible nature of the acylation reaction is not exclusively driven by the strain of the four-membered ring of β-lactams. Results Modification of the absorbance spectrum of nitrocefin following acylation of the L,D- transpeptidase Ldtfm. Hydrolysis of the β-lactam ring of nitrocefin (50 µM) by the β-lactamase BlaC (1 µM) in 100 mM sodium phosphate bufer (pH 6.0) produced the expected large increase in the absorbance at 486 nm (Fig. 2A). Under these conditions, the variation in the molar extinction coefcient (Δε) was 15,200 M−1 cm−1 (1,400 M−1 cm−1 versus 16,600 M−1 cm−1 for native and hydrolyzed nitrocefn, respectively). Incubation of Ldtfm (22 µM) with increasing concentrations of nitrocefn (0 to 56 µM) also led to an increase of the absorbance at 486 nm (Fig. 2B). Te increase in the absorbance was linear until the concentration of nitrocefn exceeded that of the enzyme. Tis result indicates that the increase in the absorbance at 486 nm is due to the rupture of SCIENTIFIC REPORTS | 7: 9136 | DOI:10.1038/s41598-017-09341-8 2 www.nature.com/scientificreports/ Figure 2. Determination by spectrophotometry of the acylation of Ldtfm by nitrocefn. (A) Absorbance spectra of various forms of nitrocefn (50 µM). Blue, native nitrocefn; orange, nitrocefn hydrolyzed by BlaC; grey, nitrocefn in the acyl-enzyme formed with Ldtfm. (B) Titration of the active site of Ldtfm (22 µM) by nitrocefn. (C and D) Mass spectra of Ldtfm and of the acyl-enzyme formed with nitrocefn. Te average mass of Ldtfm (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. Te calculated mass of nitrocefn, Ldtfm, and of the acyl-enzyme were 516.5 Da, 16,639.3 Da, and 17,155.8 Da, respectively. the β-lactam ring of nitrocefn upon acylation of the catalytic Cys residue of Ldtfm. In agreement, a covalent adduct was detected by mass spectrometry (Fig. 2C). Te spectra of nitrocefn hydrolyzed by BlaC and of nitrocefn linked to Ldtfm in the acyl-enzyme were similar with two minor diferences. Nitrocefn in the acyl-enzyme absorbed less than hydrolyzed nitrocefn (ε = 9,900 M−1 cm−1 versus 16,600 M−1 cm−1 at 486 nm, respectively). A minor red shif might occur in the absorbance maximum (λmax of 501 nm versus 486 nm, respectively). Tus, nitrocefn provides a sensitive assay to titrate the active site of Ldtfm. Kinetic analyses of Ldtfm acylation by nitrocefin. Stopped-flow kinetics were performed to assess the efcacy of acylation of Ldtfm (11 µM) by nitrocefn (25, 50, 75, and 100 µM; Fig. 3A). Te concentration of the acyl-enzyme (EI*) was determined based on the diference in the molar extinction coefcient −1 −1 −1 −1 (Δε486 nm = 8,500 M cm ) between native nitrocefn (ε = 1,400 M cm ) and nitrocefn in the acyl-enzyme −1 −1 (ε = 9,900 M cm ) (Fig. 2). To determine the value of kobs, as defned in equation 1 (Fig. 3), exponential rise to maximum was ftted to kinetic data. Full acylation of Ldtfm was observed at all the concentrations of nitrocefn that were tested and the value of kobs increased linearly with the concentration of nitrocefn (Fig. 3B). Te slope provided an estimate of the efcacy of the acylation reaction (24,000 ± 700 M−1 s−1).

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