Structural insight into the inactivation of Mycobacterium tuberculosis non-classical transpeptidase LdtMt2 by biapenem and tebipenem

Abstract

BackgroundThe carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).ResultsHere, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases.ConclusionThe results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-017-0082-4) contains supplementary material, which is available to authorized users.

Targeting the Mycobacterium tuberculosis transpeptidase LdtMt2 with cysteine-reactive inhibitors including ebselen

“…11,28 The overall fold of Ldt Mt2 in our structure aligns well with reported Ldt Mt2 structures, 11,28 with a root-mean-square-deviation of 0.70 Å for backbone Ca atoms compared to PDB entry 5D7H. 29 Ldt Mt2 crystals were soaked with ebselen, and the structure of the complex was solved by molecular replacement (Table S2, Fig. 2A and Fig.…”

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 .…”

A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

“…These representative sequences were used to build a phylogenetic tree using the maximum likelihood method (Figure 3A). Homologs of Tae5 STM clustered into 5 main clades: clade 1 (red, 6332 sequences), to which Tae5 STM belongs, is composed of proteins containing the uncharacterized DUF2778 domain (DUF2778 superfamily); clade 2 (blue, 44540 sequences) contains the L,D-transpeptidases from Bacillus subtilis (Ldt Bs , PDB 1Y7M) (Bielnicki et al, 2006); clade 3 (green, 44090 sequences) contains L,D-transpeptidases from Enterococcus faecium (Ldt fm , PDB 1ZAT) (Biarrotte-Sorin et al, 2006), Mycobacterium abscessus (LdtMa b , PDB 5UWV) (Kumar et al, 2017) and Mycobacterium tuberculosis (Ldt Mt1-3 , PDB 3TUR, 5DCC) (Erdemli et al, 2012; Bianchet et al, 2017); clade 4 (purple, 25242 sequences) contains an enzyme from Helicobacter pylori (Cds6, PDB 4XZZ) that has a catalytic domain resembling L,D-transpeptidases but with L,D-carboxypeptidase activity (Kim et al, 2015); clade 5 (gray, 25765 sequences) contains an L,D-transpeptidase from E. coli (YcbB, PDB 6NTW) (Caveney et al, 2019) and proteins recognized by the Pfam model YkuD_2.…”