Regiochemical and Stereochemical Course of the Reaction Catalyzed by the Fosfomycin Resistance Protein, FosA

Bernat, Bryan A.; Laughlin, and L. Timothy; Armstrong, Richard N.

doi:10.1021/jo980014b

Cited by 21 publications

(36 citation statements)

References 12 publications

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“…The L-cys-fosfomycin product molecule was constructed in PRODRG (28) according to the stereochemistry originally reported by Bernat et al, which was biosynthesized using the FosA enzyme (31). Thus, the resulting 2 product can be correctly identified as (1R,2S)-1-(S-L-cysteinyl)-2-hydroxypropylphosphonate, the same product as that reported for FosA (31). Inversion of configuration at C1 indicates that the reaction proceeds via direct S N 2 addition of the thiol to the oxirane carbon.…”

Section: Resultsmentioning

confidence: 99%

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

et al. 2013

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The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M2+ dependent thiol tranferases that catalyze nucleophilic addition of either L-cysteine (L-cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosBBc). The overall structure of FosBBc, at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosBBc co-crystallized with fosfomycin and a variety of divalent metals, including Ni2+, Mn2+, Co2+, and Zn2+, indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosBBc structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate L-cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosBBc in complex with the L-cysteine-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn2+ metal in the active site have been determined. The L-cysteine moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosBBc indicated that the enzyme has a preference for bacillithiol over L-cysteine when activated by Mn2+ and is inhibited by Zn2+. The fact that Zn2+ is an inhibitor of FosBBc was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-cysteine bound.

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Section: Resultsmentioning

confidence: 99%

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

et al. 2013

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“…Addition at the C1 position was unexpected as it is extremely hindered by the phosphonyl function. Indeed, the acid-catalyzed addition to fosfomycin is to the much less hindered C2 [39].…”

Section: Oxirane Ring Nucleophilic Addition Family: Fosfomycin Resistmentioning

confidence: 98%

Structural and mechanistic comparisons of the metal-binding members of the vicinal oxygen chelate (VOC) superfamily

Moran

2011

Journal of Inorganic Biochemistry

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“…Of the two, only L-cysteine supports a modest enzyme activity ( Table 2). The product of the FosB-catalyzed addition of L-cysteine to fosfomycin, examined by heteronuclear multiple-bond correlation nuclear magnetic resonance spectroscopy, is (1R,2S)-1-(S-L-cysteinyl)-2-hydroxypropylphosphonate (data not shown), identical to the product produced by FosA with L-cysteine (4). Extended incubations of FosB, fosfomycin, and various divalent metal ions indicated that no degradation of the antibiotic occurred in the absence of a thiol substrate.…”

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“…Since B. subtilis, like many gram-positive bacteria, lacks detectable levels of glutathione (5,15), it seems likely that FosB must use a different thiol. To identify the FosB thiol cofactor, the rate of appearance of conjugates of fosfomycin with various thiols was determined as described previously (3,4). L-Cysteine and coenzyme A sulfhydryl (CoASH) are two abundant thiols in gram-positive bacteria (5,15).…”

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FosB, a Cysteine-Dependent Fosfomycin Resistance Protein under the Control of ς ^W , an Extracytoplasmic-Function ς Factor in Bacillus subtilis

et al. 2001

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We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires W , and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn 2؉ -dependent glutathione transferases but with a preference for Mg 2؉ and L-cysteine as cofactors.Sequencing of the Bacillus subtilis genome revealed the presence of seven new factors, all members of the extracytoplasmic function subfamily (12, 13). We have begun to investigate their functions by mutation of each gene and the identification of target operons (8)(9)(10)(11). In this work, we demonstrate that yndN encodes a fosfomycin resistance (Fos r ) protein that depends on W for expression. We have renamed yndN as fosB, based on its similarity to the fosB gene identified on a Staphylococcus epidermidis plasmid (Fig. 1B).Transcription of fosB requires W . Previously, 15 W -dependent operons were identified by searching the genome for sequences matching the W autoregulatory site, P w : TGAAAC N 16 CGTA (10). Additional candidate promoters, including one for fosB (Fig. 1A), were identified with 17-bp spacer regions (10).To confirm the role of this predicted W -dependent promoter, we generated a P fosB -cat-lacZ operon fusion inserted ectopically in the SP␤ prophage (HB8083; Table 1) and transduced the reporter fusion into wild-type, sigW, and rsiW mutant backgrounds. Promoter activity as determined in early-stationary-phase cells yielded 18.4 Miller units of ␤-galactosidase in the wild-type strain (HB0052), and this was reduced to background levels (ϳ1 unit) in the sigW mutant (HB0023). In the rsiW (anti-W ) mutant (HB0012), expression was elevated approximately twofold (30.5 units). This pattern is precisely that expected for a W -dependent promoter. We used reverse transcriptase primer extension mapping to identify the transcriptional start site for fosB as a G residue 10 bases downstream from the Ϫ10 region CGTA motif (Fig. 2). There were no other start sites visible in the primer extension experiment, which is consistent with the idea that W is largely, if not exclusively, responsible for fosB transcription. The fosB gene is apparently monocistronic, as it is flanked on either side by genes transcribed from the complementary strand of the genome (Fig. 1A).fosB and sigW mutants are sensitive to fosfomycin. Both fosB (HB0008) and sigW (HB0020) mutants are fosfomycin sensitive: an MIC of 50 g/ml for the mutants compared to 800 g/ml for the wild type in liquid culture. Similarly, the sigW and fosB mutants have a much greater zone of growth inhibition in disk diffusion assays (ϳ25-mm zone for wild type versus Ͼ50 mm for the mutants). The fosB and sigW mutant strains did not display altered sensitivity to several other antibiotics, including vancomycin, cephalosporin C, penicillin G, D-cycloserine, tunicamycin, nisin, and bacitracin. Induction of fosB from a xylose-inducible promoter completely restores Fos r to either the sigW mutant (HB0081) or, as expected, t...

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Regiochemical and Stereochemical Course of the Reaction Catalyzed by the Fosfomycin Resistance Protein, FosA

Cited by 21 publications

References 12 publications

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

Structural and mechanistic comparisons of the metal-binding members of the vicinal oxygen chelate (VOC) superfamily

FosB, a Cysteine-Dependent Fosfomycin Resistance Protein under the Control of ς ^W , an Extracytoplasmic-Function ς Factor in Bacillus subtilis

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Regiochemical and Stereochemical Course of the Reaction Catalyzed by the Fosfomycin Resistance Protein, FosA

Cited by 21 publications

References 12 publications

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

Structural and Chemical Aspects of Resistance to the Antibiotic Fosfomycin Conferred by FosB from Bacillus cereus

Structural and mechanistic comparisons of the metal-binding members of the vicinal oxygen chelate (VOC) superfamily

FosB, a Cysteine-Dependent Fosfomycin Resistance Protein under the Control of ς W , an Extracytoplasmic-Function ς Factor in Bacillus subtilis

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FosB, a Cysteine-Dependent Fosfomycin Resistance Protein under the Control of ς ^W , an Extracytoplasmic-Function ς Factor in Bacillus subtilis