Phosphonoformate: a minimal transition state analogue inhibitor of the fosfomycin resistance protein, FosA.
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 (K(i) = 0.4 +/- 0.1 microM, K(d) approximately 0.2 microM), 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 A 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 (k(on) approximately 10(7)-10(8) M(-1) s(-1)) and an off rate (k(off) = 5 s(-1)) that is slower than that for fosfomycin (k(off) = 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.
Rigsby
Rachel E
RE
Department of Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Rife
Chris L
CL
Fillgrove
Kerry L
KL
Newcomer
Marcia E
ME
Armstrong
Richard N
RN
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D017245:Foscarnet / Q000737:chemistry*; D005578:Fosfomycin / Q000737:chemistry*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D063065:Organophosphonates / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000737:chemistry*
D001426:Bacterial Proteins; D063065:Organophosphonates; D005578:Fosfomycin; D017245:Foscarnet; D008345:Manganese; D005982:Glutathione Transferase