The fungal product terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA)

Abstract

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus. We found that terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme MurA from Enterobacter cloacae and Escherichia coli in vitro. The crystal structure of the MurA dead-end complex with terreic acid revealed that the quinine ring is covalently attached to the thiol group of Cys115, the molecular target of the antibiotic fosfomycin. Kinetic characterization established that the inactivation requires the presence of substrate UNAG (UDP-N-acetylglucosamine), proceeding with an inactivation rate constant k(inact) of 130 M(-1) s(-1). Although the mechanisms of inactivation are similar, fosfomycin is approximately 50 times more potent than terreic acid, and the structural consequences of covalent modification by these two inhibitors are fundamentally different. The MurA-fosfomycin complex exists in the closed enzyme conformation, with the Cys115-fosfomycin adduct buried in the active site. In contrast, the dead-end complex with terreic acid is open, is free of UNAG, and has the Cys115-terreic acid adduct solvent-exposed. It appears that terreic acid reacts with Cys115 in the closed, binary state of the enzyme, but that the resulting Cys115-terreic acid adduct imposes steric clashes in the active site. As a consequence, the loop containing Cys115 rearranges, the enzyme opens, and UNAG is released. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflect the importance of noncovalent binding potential, even for covalent inhibitors, in ensuring inactivation efficiency and specificity.

FIGURE 1. Reaction catalyzed by MurA

FIGURE 2. Inactivation of E. cloacae MurA by terreic acid

(A) Time-dependent loss of MurA activity in the presence of 0.05 mM UNAG. The terreic acid concentration was 0.01 mM (•), 0.02 mM (○), 0.04 mM (▼), 0.08 mM (△), 0.16 mM (■), and 0.32 mM (□). Data were fit to equation 1. (B) Replot of the observed first-order rate constants of inactivation (k_obs) vs. terreic acid at varying UNAG concentrations [0.005 mM (•), 0.01 mM (○), 0.025 mM (▼), 0.05mM (△), 0.10 mM (■)]. Data were fit to equation 2. (C) Replot of the second-order inactivation rate constants (k_inact) vs. UNAG concentration. Data were fit to equation 3, yielding k*_inact = 130 ± 5.5 M⁻¹s⁻¹ and K_d(UNAG) = 7 ± 1 µM. (D) Dose-response curves for inhibition of MurA by fosfomycin (•) and terreic acid (○). The enzyme was preincubated with inhibitor and 0.1mM UNAG for 8 minutes before the reaction was started by addition of 1mM PEP. Data were fit to equation 4, yielding IC₅₀ values of 0.25 ± 0.01 µM for fosfomycin and 14 ± 0.6 µM for terreic acid.

FIGURE 3. Crystal structure of the dead-end complex of E. cloacae MurA with terreic acid

(Top, stereoview) The overall structure of MurA inactivated by terreic acid exists in an open conformation and is free of UNAG. The loop hosting Cys115 is shown in magenta; the covalently bound terreic acid molecule (cyan) is largely solvent–exposed. (Middle, stereoview) A detailed view of the Cys115-terreic acid adduct reveals multiple polar interactions (d ≤ 3.3 Å, black dotted lines) with water molecules (orange spheres) and two Ca²⁺ ions from the crystallization solution (green spheres); hydrophobic interactions (d ≤ 3.8 Å, green dotted lines) exist with Leu138. The blue-colored mesh represents the F_o-F_c difference electron density map (at 2.25 Å resolution and contoured at 3σ), omitting the modified Cys115 residue during the refinement. (Bottom) Proposed chemical reaction of terreic acid with Cys115.

FIGURE 4. Crystal structure of the dead-end complex of E. cloacae MurA with fosfomycin

(Top, stereoview) The overall structure of MurA inactivated by fosfomycin exists in a closed conformation with UNAG bound. The loop hosting Cys115 is shown in magenta; the covalently bound fosfomycin molecule (cyan) occupies the putative PEP-binding site. (Middle, stereoview) A detailed view of the Cys115-fosfomycin adduct reveals multiple polar and electrostatic interactions (d ≤ 3.5 Å, black dotted lines) with active site residues, water molecules and the UNAG molecule. Hydrophobic interactions (d ≤ 3.8 Å, green dotted lines) exist with Ile117. The blue-colored mesh represents the F_o-F_c difference electron density map (at 1.75 Å resolution and contoured at 3σ), omitting the Cys115-fosfomycin adduct during the refinement. (Bottom) Chemical reaction of fosfomycin with Cys115.

FIGURE 5. The molecular modes of action of terreic acid and fosfomycin on E. cloacae MurA

In its unliganded state, MurA exists in an open conformation (E_open) with Cys115 solvent–exposed. Binding of the substrate UNAG (S₁) induces the structural transition to the closed binary state (E_closed:S₁). Both terreic acid and fosfomycin interact with Cys115 in this binary complex. The reaction of terreic acid with Cys115 forces the enzyme to open and to release UNAG, leading to an open dead-end complex (E_open-I). By contrast, fosfomycin interaction with the binary complex renders the overall conformation of MurA unchanged, and the dead-end complex exists as the closed enzyme state with UNAG still bound (E_closed-I). The loop containing Cys115 is shown in magenta, UNAG in yellow, and the inhibitors in cyan.