Mechanism-Based Inhibition of the Mycobacterium tuberculosis Branched-Chain Aminotransferase by d- and l-Cycloserine

Abstract

The branched-chain aminotransferase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme responsible for the final step in the biosynthesis of all three branched-chain amino acids, l-leucine, l-isoleucine, and l-valine, in bacteria. We have investigated the mechanism of inactivation of the branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) by d- and l-cycloserine. d-Cycloserine is currently used only in the treatment of multidrug-drug-resistant tuberculosis. Our results show a time- and concentration-dependent inactivation of MtIlvE by both isomers, with l-cycloserine being a 40-fold better inhibitor of the enzyme. Minimum inhibitory concentration (MIC) studies revealed that l-cycloserine is a 10-fold better inhibitor of Mycobacterium tuberculosis growth than d-cycloserine. In addition, we have crystallized the MtIlvE-d-cycloserine inhibited enzyme, determining the structure to 1.7 Å. The structure of the covalent d-cycloserine-PMP adduct bound to MtIlvE reveals that the d-cycloserine ring is planar and aromatic, as previously observed for other enzyme systems. Mass spectrometry reveals that both the d-cycloserine- and l-cycloserine-PMP complexes have the same mass, and are likely to be the same aromatized, isoxazole product. However, the kinetics of formation of the MtIlvE d-cycloserine-PMP and MtIlvE l-cycloserine-PMP adducts are quite different. While the kinetics of the formation of the MtIlvE d-cycloserine-PMP complex can be fit to a single exponential, the formation of the MtIlvE l-cycloserine-PMP complex occurs in two steps. We propose a chemical mechanism for the inactivation of d- and l-cycloserine which suggests a stereochemically determined structural role for the differing kinetics of inactivation. These results demonstrate that the mechanism of action of d-cycloserine's activity against M. tuberculosis may be more complicated than previously thought and that d-cycloserine may compromise the in vivo activity of multiple PLP-dependent enzymes, including MtIlvE.

Figure 1

Mechanism-based inhibition of MtIlvE by (A) DCS and (B) LCS. (A) ln % of remaining activity of MtIlvE inhibited over time and increasing concentrations of DCS as follows: (⧫) control, (▼) 1 mM, (▲) 2 mM, (■) 3 mM, and (●) 4 mM. (B) ln % of remaining activity of MtIlvE inhibited over time and increasing concentrations of LCS as follows: (⧫) control, (●) 25 μM, (■) 50 μM, (▲) 75 μM, and (▼) 100 μM. The insets represent the Kitz-Wilson replots and describe the t_1/2 life. The second-order rate constant k_inact/K_I is calculated as 1/slope.

Figure 2

ESI-MS analysis of intact molecular weights of MtIlvE-PLP inhibited overnight by (A) DCS and (B) LCS. The peak at m/z 332.06 represents, in both cases, the presence of a noncovalent protein inhibitor complex where the cycloserine ring is intact.

Figure 3

UV–vis analysis of MtIlvE inhibition by the cycloserine isomers over time. The upper blue lines on (A) and (B) represent MtIlvE-PLP in the absence of inhibitor. All the other lines represent the spectra of MtIlvE in the presence of (A) 1 mM DCS at 1, 6, 12, 15, 25, 35, 45, 60, 90, 120, 180, and 300 min, respectively. The inset in (A) represents the change in absorbance at λ_max ≈ 415 nm over time. The data was fit to a single exponential equation; and (B) 0.5 mM LCS at 1, 6, 25, 45, 90, 120, 180, 240, 300, 360, 420, 600, 1050, and 1665 min, respectively. The two lower panels represent the change in absorbance at λ_max ≈ 415 nm over time. The data on the left lower panel was fit to a single exponential equation. The right lower panel was fit to a double exponential equation. In both (A) and (B), the peak at λ_max ≈ 415 nm (internal aldimine) is reduced over time, while the PMP (λ_max ≈ 330 nm) peak is increased. The appearance of the PMP in the DCS inhibited enzyme happens approximately 5-fold faster than with LCS.

Figure 4

Overall structure of MtIlvE homodimer in complex with PMP-DCS. The two domains of MtIlvE are colored in orange (residues 34–173 and 363–368) and green (residues 182–362), respectively. An α-helix (residues 340–354, colored in blue), as well as a flexible loop (residues 173–182, poorly resolved in this structure), connect the two domains. The PLP-cycloserine molecule is rendered as sticks colored by CPK, with carbon atoms in white. A red circle highlights the presence of a disulfide bond covalently linking the two monomers. Close-up view (inset) of the interchain disulfide bond observed in the MtIlvE-PMP-DCS structure. The cysteines C196 are involved in the formation of a disulfide bond located at the homodimer interface. The electron density of a 2Fo-Fc map (blue) is shown contoured at 1σ. The two residues are represented as sticks colored by CPK, with carbon and sulfur atoms colored in green and yellow, respectively.

Figure 5

Active site of the MtIlvE-PLP-DCS structure. (A) Superimposition of the MtIlvE-PMP (orange) and MtIlvE-PLP-DCS (green) structures. Residues interacting directly with either PMP or PLP-DCS adduct are representing as sticks colored by CPK whereas the water molecules are rendered as spheres. When two water molecule positions overlap, the first number corresponds to the MtIlvE-PLP-DCS structure and the second to the MtIlvE-PMP structure. W423 was replacing the cycloserine ring in the MtIlvE-PMP structure. (B,C) The electron density of a Fo-Fc omit map (blue) is shown contoured at 3σ, the PLP-cycloserine molecule was omitted during map calculation. PLP-cycloserine is represented as sticks colored by CPK, with carbon atoms in yellow. The residues participating in hydrogen bonding (dashes) with PLP-cycloserine are rendered as sticks, while the water molecules (W) interacting with the ligand are represented as spheres. The distances are given in angstroms.

Scheme 1

Proposed Aromatization Mechanism of Inactivation of MtIlvE by LCS (left) and DCS (right)