Inactivation of Mycobacterium tuberculosis l,d-transpeptidase LdtMt₁ by carbapenems and cephalosporins

Abstract

The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase Ldt(Mt1) by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt(Mt1), which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt(Mt1), with a binding rate constant of 0.08 μM(-1) min(-1). The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min(-1)) in comparison to the acylation rate constant (3.1 min(-1)). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10(-3) min(-1). Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt(Mt1) inactivator. Cephalosporins also formed covalent adducts with Ldt(Mt1), although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.

Fig 1

Reaction catalyzed by peptidoglycan transpeptidases. (A) Formation of 3→3 cross-links by l,d-transpeptidases (Ldt). Cross-links are indicated by double arrows. G, N-acetylglucosamine; M, N-glycolyl-muramic acid; mDap, meso-diaminopimelic acid; mDap_NH2, mDap with an amidated ε carboxyl group; D-iGln, d-iso-glutamine. (B) Formation of 4→3 peptidoglycan cross-links by classical d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family. (C) Ldt acylation by carbapenems. (D) Full catalytic cycle. Binding of a β-lactam (I) to the enzyme (E) leads to formation of a noncovalent complex (EI). The chemical step of the reaction leads to formation of an acylenzyme (EI*), which is slowly hydrolyzed to produce free enzyme and hydrolyzed β-lactam (I_hydrol). Dashed arrows indicate slow reactions.

Fig 2

Fluorescence kinetics of Ldt_Mt1 inactivation by carbapenems. (A) Simulation of variations in the concentrations of the three enzyme forms. The initial concentrations of enzyme (E = E_total at time zero) and inhibitor (I = I_total at time zero) were 5 and 100 μM, respectively. The values 0.08 μM⁻¹ min⁻¹, 0.1 min⁻¹, and 3.1 min⁻¹ were attributed to catalytic constants k₁, k₋₁, and k_inact, respectively. (B) Relative fluorescence intensities of the three enzyme forms. (C) Simulation of fluorescence kinetics. The black curve was computed with the kinetic constants and fluorescence intensities used for the data shown in panels A and B. The red and blue curves show the impact of 2-fold decreases in k₁, and k_inact, respectively. The gray curve corresponds to experimental data. (D) Determination of kinetic constants for meropenem, doripenem, imipenem, and ertapenem. The values of k₁ and k_inact were determined by fitting simulations (solid lines) to experimental data for drug concentrations of 100 μM (green) and 300 μM (blue).

Fig 3

Ldt_Mt1 inactivation by carbapenems. (A) Structures of carbapenems. (B) Masses (atomic mass units) of covalent adducts resulting from acylation of Ldt_Mt1 by carbapenems. Increments were obtained by subtracting the observed mass of the native enzyme from that of acylenzymes. (C) Determination of the rate constant for acylenzyme hydrolysis (k_hydrol) for meropenem (blue), doripenem (green), imipenem (purple), and ertapenem (red) (values are fit ± standard deviation [SD]). Rates of hydrolysis were deduced from the slopes. V, hydrolysis velocity; ND, not detected.

Fig 4

Ldt_Mt1 inactivation by cephalosporins. (A) Structure of cephalosporins with common R₁ (wheat) and R₂ (blue) side chains highlighted. (B) Average mass (atomic mass units) of acylenzymes. (C) Kinetics of Ldt_Mt1 inactivation by cephalothin (blue), cefotaxime (green), and ceftriaxone (red) (values are fit ± SD). The slope provides an estimate of the ratio of k_inact over K_app. (D) Kinetics of acylenzyme hydrolysis. The slope provides an estimate of the rate constant k_hydrol for hydrolysis of acylenzymes formed with cephalothin (blue), cefotaxime (green), and ceftriaxone (red) (values are fit ± SD).

Fig 5

Reactions of Ldt_Mt1 with cephalosporins (A) and carbapenems (B).