53 research outputs found
Structure and mechanism of the genomically encoded fosfomycin resistance protein, FosX, from Listeria monocytogenes
The fosfomycin resistance protein, FosX, catalyzes the hydration of the antibiotic fosfomycin, (1R,25)-epoxypropylphosphonic acid. Genes encoding the enzyme are found in several pathogenic microorganisms. The structure and mechanism of action of the genomically encoded FosX enzyme from Listeria monocytogenes (FosXLMATCC) obtained from the American Type Culture Collection are reported. The gene harbors 31 point mutations, and as a consequence, the protein differs in 10 amino acid residues from the previously reported FosX encoded in the genome of the EGD strain of L. monocytogenes (FosXLMEGD). The FosXLMATCC enzyme is shown to catalyze the addition of water to the C1 position of the antibiotic with inversion of configuration at C1. The reaction involves Mn(II) activation of the oxirane oxygen and E44 acting as a general base. The structure of the enzyme has been determined from six different crystal forms of the protein. The structures of the enzyme without metal bound are similar but differ in the loop regions. Perhaps the most informative structure is the one with the product bound. This structure shows that the phosphonate group of the product is bound in an orientation that is different than that of fosfomycin bound to the related enzyme, FosA. The implication is that the substrate may also be bound in a different orientation in FosX. A high-resolution structure (1.44 Å resolution) of the enzyme reveals a unique conformation in which the C-terminal tail of the protein coordinates to the Mn(II) center via the carboxylate of E126. The kinetic characterization of the E126Q mutant indicates that this conformation of the protein is probably not relevant to the function of the enzyme. Kinetic analysis of mutants of active site residue E44 is consistent with its proposed roll as a general base catalyst in the addition of water to the antibiotic. © 2007 American Chemical Society
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 (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
A genome-wide analysis of Escherichia coli responses to fosfomycin using TraDIS-Xpress reveals novel roles for phosphonate degradation and phosphate transport systems
BACKGROUND: Fosfomycin is an antibiotic that has seen a revival in use due to its unique mechanism of action and efficacy against isolates resistant to many other antibiotics. In Escherichia coli, fosfomycin often selects for loss-of-function mutations within the genes encoding the sugar importers, GlpT and UhpT. There has, however, not been a genome-wide analysis of the basis for fosfomycin susceptibility reported to date. METHODS: Here we used TraDIS-Xpress, a high-density transposon mutagenesis approach, to assay the role of all genes in E. coli involved in fosfomycin susceptibility. RESULTS: The data confirmed known fosfomycin susceptibility mechanisms and identified new ones. The assay was able to identify domains within proteins of importance and revealed essential genes with roles in fosfomycin susceptibility based on expression changes. Novel mechanisms of fosfomycin susceptibility that were identified included those involved in glucose metabolism and phosphonate catabolism (phnC-M), and the phosphate importer, PstSACB. The impact of these genes on fosfomycin susceptibility was validated by measuring the susceptibility of defined inactivation mutants. CONCLUSIONS: This work reveals a wider set of genes that contribute to fosfomycin susceptibility, including core sugar metabolism genes and two systems involved in phosphate uptake and metabolism previously unrecognized as having a role in fosfomycin susceptibility
Antibiotherapy and pathogenesis of uncomplicated UTI: difficult relationships
In a time when conventional antibiotics are becoming increasingly less effective for treatment of infections, the relationship between bacteria and antimicrobial resistance is becoming more and more complicated. This paper provides a current review of studies reported in the literature pertaining to the antibiotherapy of human urinary tract infections (UTI), in a way that helps the reader direct a bibliographic search and develop an integrated perspective of the subject. Highlights are given to (bio)pathogenesis of uncomplicated cystitis. Features associated with the antibiotherapy of UTI such as development of resistance are presented in the text systematically. This review discusses recent advances in the understanding of how the predominant uropathogen Escherichia coli interacts with its host and leads to infection; so one can understand some of the reasons behind antibiotherapy failures
Determinants of Regioselectivity and Chemoselectivity in Fosfomycin Resistance Protein FosA from QM/MM Calculations
The Mechanism of Dienoyl-CoA Reduction by 2,4-Dienoyl-CoA Reductase Is Stepwise: Observation of a Dienolate Intermediate â€
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New high-capacity, calcium-based sorbents, calcium silicate sorbents. Final report, 1993--August 31, 1994
A search is being carried out for new calcium-based S0{sub 2} sorbents for induct injection. More specifically, a search is being carried out for induct injection calcium silicate sorbents that are highly cost effective. The objectives for the current year include the study of sorbents made from Ca(OH){sub 2}, from mixtures of Ca(OH){sub 2} and SiO{sub 2}, and from portland cement. They also include the study of sorbents made from model compounds. During this year, sorbents prepared from Ca(OH){sub 2} and from mixtures of Ca(OH){sub 2} and fumed SiO{sub 2} were investigated. The results show that very good SiO{sub 2}-modified Ca(OH){sub 2} sorbents in which the Si-to-Ca reactant ratio is low can be prepared from Ca(OH){sub 2} and fumed SiO{sub 2}. Sorbents prepared from Ca(OH){sub 2} and natural SiO{sub 2} or natural SiO{sub 2} sources were also studied. The results obtained show that very good SiO{sub 2}-modified Ca(OH){sub 2} sorbents and calcium silicate hydrate sorbents, C-S-H sorbents, can be prepared from Ca(OH){sub 2} and diatomite, pumice or perlite, minerals that are readily available. In addition. sorbents prepared from Ca{sub 3}SiO{sub 5} and {beta}-Ca{sub 2}SiO{sub 4} and from mixtures of these compounds and SiO{sub 2} were studied. The results secured demonstrate that very good C-S-H rich sorbents can be prepared from these compounds and from mixtures of them with SiO{sub 2}. They also provide information useful for interpreting the cement sorbent results. Sorbents prepared from cement and from mixtures of cement and natural SiO{sub 2} or SiO{sub 2} sources were investigated as well. The results secured show that cement and mixtures of it with diatomite, pumice or perlite rapidly yield excellent sorbents with the proper reaction conditions
Orientation of Coenzyme A Substrates, Nicotinamide and Active Site Functional Groups in (Di)enoyl−coenzyme A Reductases â€
Evolution of the Antibiotic Resistance Protein, FosA, Is Linked to a Catalytically Promiscuous Progenitor
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