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

Abstract

Background: The 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).

Results: Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (Ldt_Mt2) 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 Ldt_Mt2. 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.

Conclusion: The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of Ldt_Mt2, 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.

Keywords: Biapenem; Carbapenem; Enzyme inactivation; L,D-transpeptidase; Mycobacterium tuberculosis; Peptidoglycan; Tebipenem.

Fig. 1

Apo-Ldt_Mt2 and adduct structures. a Overlap of the apo- (brown), biapenem- (pink), and tebipenem-complexes (cyan) of Ldt_Mt2. BlgA (aa 56–150) and BlgB (aa 150–250) are Bacterial Immunoglobulin-like (BIg) domains and CD (aa 251–408) is the catalytic domain. b & c Simulating annealing omit map at the active site of Ldt_Mt2-biapenem (b) and Ldt_Mt2-tebipenem (c). The refined structure of the respective catalytic site and adduct are shown. The adduct atoms were omitted during a mock refinement cycle including a torsional simulated annealing step using the refinement program PHENIX. The omit maps were contoured at 3.5 σ level. In both panels the MTOA-adducts are colored green (stereo views in Additional file 1). d Chemical structure of the common β-lactam core of the anticipated non-degraded adducts. e Chemical structure of the observed adduct in both complexes

Fig. 2

Adducts bound to the outer cavity of the Ldt_Mt2 catalytic site. a Overlay of the Ldt_Mt2-biapenem (enzyme carbon atoms colored in blue) and Ldt_Mt2-tebipenem (in orange) catalytic sites as viewed from the outer cavity. The protein residues that interact with the MTOA adducts are shown and common hydrogen bond interactions indicated as cyan dashed lines. b Similar view of the catalytic site of the tebipenem complex crystal structure showing a portion of the solvent accessible surface of the outer cavity and tunnel connecting to the inner cavity, displaying the adduct and residues of the Ldt motif participating in the interaction with the it. The MTOA adduct is colored green

Fig. 3

Heat exchange during biapenem and tebipenem adduct formation with Ldt_Mt2. Both carbapenem show exothermic heat exchanges after the addition of the carbapenem to the reaction cell containing enzyme

Fig. 4

Structural comparison of Mtb Ldts with different carbapenem adducts. a Overlay of catalytic domains of apo-Ldt_Mt1 (colored blue) and apo-Ldt_Mt2 (colored cyan). Red arrows show the displacement of the β-hairpin flap between paralogs. b Two views related by a counterclockwise 90° rotation of the overlay of outer cavity-bound adducts of Ldt_Mt1-imipenem (4VYM; carbon atoms of enzyme and adduct are colored blue and green, respectively) and Ldt_Mt2-MTOA corresponding to the biapenem/Ldt_Mt2 complex structure (colored in cyan and magenta). c Overlay of the Ldt_Mt2 catalytic domains of Ldt_Mt2-meropenem (3VYO; magenta) and Ldt_Mt2-MTOA adduct (cyan). Red arrows show the displacement of the β-hairpin flap between these complexes forming inner- and outer-cavity adducts, respectively. d Overlay of the catalytic site of Ldt_Mt2-meropenem (carbon atoms of the enzyme and the adduct are colored orange and green, respectively) and Ldt_Mt2-MTOA (cyan and magenta). Red arrows in the panels (b) and (d) show the displacement of the conserved Tyr³¹⁸ on the β-hairpin flap that in Ldt_Mt1-imipenem and Ldt_Mt2-meropenem interacts with the carbapenems hydroxyacetyl substituent, such interaction is loss in MTOA by the elimination of this group after adduct formation

Fig. 5

Predicted binding models of the biapenem and tebipenem interactions with Ldt_Mt2. Docking results of the binding of intact (a) biapenem and (b) tebipenem to the outer cavity of Ldt_Mt2. Left panels of (a) and (b) show the solvent accessible surface corresponding to the outer cavity and tunnel connecting to the inner cavity. The carbapenems (biapenem carbon atoms are yellow; tebipenem carbon atoms are orange) and Ldt_Mt2 residues that participate in binding (cyan). The protein-ligand interactions are shown in the right panels. Residues circled are in Van der Waals contact with the ligand; those colored green and pink are hydrophobic and hydrophilic residues, respectively. Hydrogen bonds are marked as black dashed arrows starting in the proton donor. The red dashed arrow highlights the Cys³⁵⁴-C7 tether used for the steered docking simulation. Purple clouds around carbapenem atoms indicate solvent exposure, and the size of the clouds indicates the degree of exposure. Offset blue circles indicate partial exposure of the protein residue. The drawing and analysis were performed using MOE

Fig. 6

The different accessibilities of the thioester-bond in the two observed binding modes. a View from the outer cavity of the meropenem-adduct of Ldt_Mt2 [20]. b View from the inner cavity of the MTOA-adduct of Ldt_Mt2

Fig. 7

Proposed mechanisms of biapenem and tebipenem adduct formation and degradation. The figure shows the chemical steps (steps 1–10) to reach the crystallographically observed adducts from the expected initial (intact) carbapenem adduct of the enzyme. The expected mass differences in each step of the reaction are annotated for comparison with the observed species in the mass spectrometry experiments. A final decarboxylation step (step 11), where the product is observed in the ESI mass spectra but not in the crystal, is included as a possible explanation. The mass increases caused by the adducts were calculated using the program ChemDraw®(v 15.0.0.106; 1985-2015 PerkinElmer Informatics, Inc.)