Binding and processing of β‐lactam antibiotics by the transpeptidase Ldt_Mt2 from Mycobacterium tuberculosis

Abstract

b-lactam antibiotics represent a novel direction in the chemotherapy of tuberculosis that brings the peptidoglycan layer of the complex mycobacterial cell wall in focus as a therapeutic target. Peptidoglycan stability in Mycobacterium tuberculosis, especially during infection, relies on the nonconventional peptide cross-links formed by L,D-transpeptidases. These enzymes are known to be inhibited by b-lactams, primarily carbapenems, leading to a stable covalent modification at the enzyme active site. A panel of 16 b-lactam antibiotics was characterized by inhibition kinetics, mass spectrometry, and x-ray crystallography to identify efficient compounds and study their action on the essential transpeptidase, Ldt Mt2 . Members of the carbapenem class displayed fast binding kinetics, but faropenem, a penem type compound showed a three to four time higher rate in the adduct formation. In three cases, mass spectrometry indicated that carbapenems may undergo decarboxylation, while faropenem decomposition following the acylation step results in a small 87 Da b-OH-butyryl adduct bound at the catalytic cysteine residue. The crystal structure of Ldt Mt2 at 1.54A resolution with this fragment bound revealed that the protein adopts a closed conformation that shields the thioester bond from the solvent, which is in line with the high stability of this dead-end complex observed also in biochemical assays.

A Fluorescence‐Based Assay for Screening β‐Lactams Targeting the Mycobacterium tuberculosis Transpeptidase LdtMt2

“…Ldt Mt2 from Mycobacterium tuberculosis appears to be of particular importance for virulence, as its loss leads to altered morphology and inhibition of colony growth . Certain β‐lactam antibiotics inhibit Ldt Mt2 , in particular members of the (carba)penem subclass, and these represent potential leads for treatment of TB . However, inhibitor discovery and development is severely limited by the current inhibition assays used for the Ldts.…”

“…Previously described low‐throughput assays for the Ldts have relied on methods such as mass spectrometry (MS), isothermal titration calorimetry (ITC), stopped‐flow fluorescence spectroscopy and hydrolysis of the chromophore‐containing β‐lactam nitrocefin . In addition, as the Ldt Mt2 construct used for assays contains only one cysteine residue (i.e., Cys354, which is located in the active site, and is catalytically essential), the thiol‐reactive compound 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB or Ellman's reagent) has been applied in colorimetric assays . Although potentially useful, these techniques are accompanied by limitations such as poor sensitivity and high protein requirements .…”

“…In addition, as the Ldt Mt2 construct used for assays contains only one cysteine residue (i.e., Cys354, which is located in the active site, and is catalytically essential), the thiol‐reactive compound 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB or Ellman's reagent) has been applied in colorimetric assays . Although potentially useful, these techniques are accompanied by limitations such as poor sensitivity and high protein requirements . We were therefore interested in exploring the development of a high‐throughput fluorescence‐based assay for efficient screening of Ldt Mt2 inhibitors.…”

“…Inspired by the DTNB method, we considered the possibility of developing an assay based on the use of cysteine‐selective fluorogenic probes. With such an assay, the impact of inhibitors on the availability of the catalytic site could be tested through the (irreversible) reaction of the active‐site cysteine with a fluorogenic probe, providing a “nonclassical” inhibition assay.…”

The structure of the N‐terminal module of the cell wall hydrolase RipA and its role in regulating catalytic activity

“…The accessibility of free Cys383 sulfhydryl group using 5,5'‐dithio‐bis‐(2‐nitrobenzoic acid) (DTNB, Ellmann's reagent) in the active site was monitored by formation of the enzyme‐TNB complex. The assay was performed as previously described in 0.1 M potassium‐phosphate pH 8.0, 1 mM EDTA in a total volume of 50 µL, using a protein concentration of 40 µM that results in an absorbance value of about 0.15‐0.2. The protein samples were mixed with 12 µL of DTNB reagent (0.1 M potassium‐phosphate pH 8.0, 1 mM EDTA, 10 mM DTNB) and the absorption at 412 nm was recorded after 10 min incubation at 22°C.…”