Phosphonoformate: A minimal transition state analogue inhibitor of the fosfomycin resistance protein, FosA

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Fosfomycin [(1R,2S)-epoxypropylphosphonic acid] is a simple phosphonate found to have antibacterial activity against both Gram-positive and Gram-negative microorganisms. Early resistance to the clinical use of the antibiotic was linked to a plasmid-encoded resistance protein, FosA, that catalyzes the addition of glutathione to the oxirane ring, rendering the antibiotic inactive. Subsequent studies led to the discovery of a genomically encoded homologue in the pathogen Pseudomonas aeruginosa. The proteins are Mn(II)-dependent enzymes where the metal is proposed to act as a Lewis acid stabilizing the negative charge that develops on the oxirane oxygen in the transition state. Several simple phosphonates, including the antiviral compound phosphonoformate (Ki = 0.4 ± 0.1 μM, Kd ≈ 0.2 μM), are shown to be inhibitors of FosA. The crystal structure of FosA from P. aeruginosa with phosphonoformate bound in the active site has been determined at 0.95 Å resolution and reveals that the inhibitor forms a five-coordinate complex with the Mn(II) center with a geometry similar to that proposed for the transition state of the reaction. Binding studies show that phosphonoformate has a near-diffusion-controlled on rate (kon ≈ 107-108 M-1 s-1) and an off rate (koffi = 5 s-1) that is slower than that for fosfomycin (koff = 30 s-1). Taken together, these data suggest that the FosA-catalyzed reaction has a very early transition state and phosphonoformate acts as a minimal transition state analogue inhibitor.

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