Tryptophan Fluorescence Quenching in β-Lactam-Interacting Proteins Is Modulated by the Structure of Intermediates and Final Products of the Acylation Reaction

Abstract

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,Dtranspeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.Peptidoglycan is an essential constituent of bacterial cell walls since it prevents cell swelling and lysis by mechanically sustaining the osmotic pressure of the cytoplasm 1 . This protective function depends upon synthesis and maintenance during the entire cell cycle of the net-like peptidoglycan macromolecule, which completely surrounds the bacterial cell. Peptidoglycan is made of glycan strands cross-linked by short peptide stems. In most bacteria, the cross-linking step is performed by D,Dtranspeptidases, which are the essential targets of β-lactam antibiotics and are often referred to as penicillin-binding proteins (PBPs) 2 . In mycobacteria [3][4] and in Clostridium difficile [5][6] , the cross-links found are mainly (70% to 80%) formed by a second class of enzymes, the L,D-transpeptidases (LDTs). Since LDTs are not inhibited by β-lactams belonging to the penam class, such as ampicillin, 7-8 , these enzymes are responsible for high-level resistance to these drugs in mutants of Enterococcus faecium 9-10 and Escherichia coli 11 selected in laboratory conditions.PBPs and LDTs are structurally unrelated [12][13][14][15] and proceed through different catalytic mechanisms for activation of Ser and Cys nucleophiles 16 , which are part of Lys-Ser 17 and Cys-His-Asp 18...

Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross‐Linking Activity of a Transpeptidase of l,d Specificity

“… Two‐step acylation of l,d ‐transpeptidase Ldt fm by carbapenems. The evidence for the formation of the amine anion in the first step of the acylation reaction is supported by previous studies [8, 43, 44] …”

“…This first step was proposed to lead to reversible formation of an amine anion [41] . The second step is irreversible, except for the cephalosporin nitrocefin, owing to rupture of the β‐lactam C−N bond followed by protonation of the nitrogen in the case of carbapenems [42, 43] . Mass spectrometry analyses showed that all carbapenems synthesized in this study formed the expected acyl enzymes without any secondary modification of the compound following the acylation step, as is the case for other classes of β‐lactams (Table 2).…”

“…[41] The second step is irreversible, except for the cephalosporin nitrocefin, owing to rupture of the b-lactamC ÀNb ond followed by protonation of the nitrogen in the case of carbapenems. [42,43] Mass spectrometry analyses showed that all carbapenems synthesized in this study Table1. Synthesis of peptido-carbapenems 28 a-n.…”

Click and Release Chemistry for Activity‐Based Purification of β‐Lactam Targets

“…These results indicate that we have successfully functionalized the carbapenem core without impairing the efficiency of Ldt fm acylation or the stability of the resulting adduct. [29,30] Capture of Ldt fm and release of the fluorescent protein adduct CBA-2 was used to explore the efficacy of step 3 (capture) and step 4 (release) of our click and release strategy (Figure 2). Ldt fm (0.65 nmol) was acylated with 6 equivalents of CBA-2 (Figure 2, step 1).…”

“…(d) Relative fluorescence of the three forms of Ldt fm . Biphasic fluorescence kinetics are accounted for by the low fluorescence intensity of the amine anion intermediate [29,30].…”