PMC

(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 .
(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 .
(C) Replot of the second-order inactivation rate constants (k_inact) vs. UNAG concentration. Data were fit to equation , 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 , yielding IC₅₀ values of 0.25 ± 0.01 µM for fosfomycin and 14 ± 0.6 µM for terreic acid.

Structures of MurA inhibitors fosfomycin (1) and terreic acid (2).

(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.

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.