Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion.

IMPORTANCE: Society uses thousands of organofluorine compounds, sometimes denoted per- and polyfluoroalkyl substances (PFAS), in hundreds of products, but recent studies have shown some to manifest human and environmental health effects. As a class, they are recalcitrant to biodegradation, partly due to the paucity of fluorinated natural products to which microbes have been exposed. Another limit to PFAS biodegradation is the intracellular toxicity of fluoride anion generated from C-F bond cleavage. The present study identified a broader substrate specificity in an enzyme originally studied for its activity on the natural product fluoroacetate. A recombinant Pseudomonas expressing this enzyme was used here as a model system to better understand the limits and effects of a high level of intracellular fluoride generation. A fluoride stress response has evolved in bacteria and has been described in Pseudomonas spp. The present study is highly relevant to organofluorine compound degradation or engineered biosynthesis in which fluoride anion is a substrate.

Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases.

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.

Biodegradation of Amphipathic Fluorinated Peptides Reveals a New Bacterial Defluorinating Activity and a New Source of Natural Organofluorine Compounds.

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.

Succinate prodrugs as treatment for acute metabolic crisis during fluoroacetate intoxication in the rat.

Sodium fluoroacetate (FA) is a metabolic poison that systemically inhibits the tricarboxylic acid (TCA) cycle, causing energy deficiency and ultimately multi-organ failure. It poses a significant threat to society because of its high toxicity, potential use as a chemical weapon and lack of effective antidotal therapy. In this study, we investigated cell-permeable succinate prodrugs as potential treatment for acute FA intoxication. We hypothesized that succinate prodrugs would bypass FA-induced mitochondrial dysfunction, provide metabolic support, and prevent metabolic crisis during acute FA intoxication. To test this hypothesis, rats were exposed to FA (0.75 mg/kg) and treated with the succinate prodrug candidate NV354. Treatment efficacy was evaluated based on cardiac and cerebral mitochondrial respiration, mitochondrial content, metabolic profiles and tissue pathology. In the heart, FA increased concentrations of the TCA metabolite citrate (+ 4.2-fold, p < 0.01) and lowered ATP levels (- 1.9-fold, p < 0.001), confirming the inhibition of the TCA cycle by FA. High-resolution respirometry of cardiac mitochondria further revealed an impairment of mitochondrial complex V (CV)-linked metabolism, as evident by a reduced phosphorylation system control ratio (- 41%, p < 0.05). The inhibition of CV-linked metabolism is a novel mechanism of FA cardiac toxicity, which has implications for drug development and which NV354 was unable to counteract at the given dose. In the brain, FA induced the accumulation of ß-hydroxybutyrate (+ 1.4-fold, p < 0.05) and the reduction of mitochondrial complex I (CI)-linked oxidative phosphorylation (OXPHOSCI) (- 20%, p < 0.01), the latter of which was successfully alleviated by NV354. This promising effect of NV354 warrants further investigations to determine its potential neuroprotective effects.

Fluoroacetate distribution, response to fluoridation, and synthesis in juvenile Gastrolobium bilobum plants.

Like angiosperms from several other families, the leguminous shrub Gastrolobium bilobum R.Br. produces and accumulates fluoroacetate, indicating that it performs the difficult chemistry needed to make a C-F bond. Bioinformatic analyses indicate that plants lack homologs of the only enzymes known to make a C-F bond, i.e., the Actinomycete flurorinases that form 5'-fluoro-5'-deoxyadenosine from S-adenosylmethionine and fluoride ion. To probe the origin of fluoroacetate in G. bilobum we first showed that fluoroacetate accumulates to millimolar levels in young leaves but not older leaves, stems or roots, that leaf fluoroacetate levels vary >20-fold between individual plants and are not markedly raised by sodium fluoride treatment. Young leaves were fed adenosine-13C-ribose, 13C-serine, or 13C-acetate to test plausible biosynthetic routes to fluoroacetate from S-adenosylmethionine, a C3-pyridoxal phosphate complex, or acetyl-CoA, respectively. Incorporation of 13C into expected metabolites confirmed that all three precursors were taken up and metabolized. Consistent with the bioinformatic evidence against an Actinomycete-type pathway, no adenosine-13C-ribose was converted to 13C-fluoroacetate; nor was the characteristic 4-fluorothreonine product of the Actinomycete pathway detected. Similarly, no 13C from acetate or serine was incorporated into fluoroacetate. While not fully excluding the hypothetical pathways that were tested, these negative labeling data imply that G. bilobum creates the C-F bond by an unprecedented biochemical reaction. Enzyme(s) that mediate such a reaction could be of great value in pharmaceutical and agrochemical manufacturing.

In vitro assessment of the dermal penetration potential of sodium fluoroacetate using a formulated product.

This paper presents experimental data on the skin absorption of sodium fluoroacetate from a formulated product using an in vitro approach and human skin. Sodium fluoroacetate is a pesticide, typically applied in formulation (1080) for the control of unwanted vertebrate invasive species. It has been assigned a Skin Notation by the ACGIH, and other international workplace health regulatory bodies, due to its predicted ability to permeate intact and abraded human skin. However, there is a distinct lack of experimental data on the skin absorption of sodium fluoroacetate to support this assignment. This study found that sodium fluoroacetate, as a formulated product, permeated the human epidermis when in direct contact for greater than 10 hr. A steady-state flux (Jss) of 1.31 ± 0.043 µg/cm2/hr and a lag time of 6.1 hr was calculated from cumulative skin permeation data. This study provides important empirical evidence in support of the assignment of a Skin Notation.

Functional characterisation of the transcriptome from leaf tissue of the fluoroacetate-producing plant, Dichapetalum cymosum, in response to mechanical wounding.

Dichapetalum cymosum produces the toxic fluorinated metabolite, fluoroacetate, presumably as a defence mechanism. Given the rarity of fluorinated metabolites in nature, the biosynthetic origin and function of fluoroacetate have been of particular interest. However, the mechanism for fluorination in D. cymosum was never elucidated. More importantly, there is a severe lack in knowledge on a genetic level for fluorometabolite-producing plants, impeding research on the subject. Here, we report on the first transcriptome for D. cymosum and investigate the wound response for insights into fluorometabolite production. Mechanical wounding studies were performed and libraries of the unwounded (control) and wounded (30 and 60 min post wounding) plant were sequenced using the Illumina HiSeq platform. A combined reference assembly generated 77,845 transcripts. Using the SwissProt, TrEMBL, GO, eggNOG, KEGG, Pfam, EC and PlantTFDB databases, a 69% annotation rate was achieved. Differential expression analysis revealed the regulation of 364 genes in response to wounding. The wound responses in D. cymosum included key mechanisms relating to signalling cascades, phytohormone regulation, transcription factors and defence-related secondary metabolites. However, the role of fluoroacetate in inducible wound responses remains unclear. Bacterial fluorinases were searched against the D. cymosum transcriptome but transcripts with homology were not detected suggesting the presence of a potentially different fluorinating enzyme in plants. Nevertheless, the transcriptome produced in this study significantly increases genetic resources available for D. cymosum and will assist with future research into fluorometabolite-producing plants.

Successful management of hyperammonemia with hemodialysis on day 2 during 5-fluorouracil treatment in a patient with gastric cancer: a case report with 5-fluorouracil metabolite analyses.

PURPOSE: Hyperammonemia is an important adverse event associated with 5-fluorouracil (5FU) from 5FU metabolite accumulation. We present a case of an advanced gastric cancer patient with chronic renal failure, who was treated with 5FU/leucovorin (LV) infusion chemotherapy (2-h infusion of LV and 5FU bolus followed by 46-h 5FU continuous infusion on day 1; repeated every 2 weeks) and developed hyperammonemia, with the aim of exploring an appropriate hemodialysis (HD) schedule to resolve its symptoms. METHODS: The blood concentrations of 5FU and its metabolites, α-fluoro-ß-alanine (FBAL), and monofluoroacetate (FA) of a patient who had hyperammonemia from seven courses of palliative 5FU/LV therapy for gastric cancer were measured by liquid chromatography-mass spectrometry. RESULTS: On the third day of the first cycle, the patient presented with symptomatic hyperammonemia relieved by emergency HD. Thereafter, the 5FU dose was reduced; however, in cycles 2-4, the patient developed symptomatic hyperammonemia and underwent HD on day 3 for hyperammonemia management. In cycles 5-7, the timing of scheduled HD administration was changed from day 3 to day 2, preventing symptomatic hyperammonemia. The maximum ammonia and 5FU metabolite levels were significantly lower in cycles 5-7 than in cycles 2-4 (NH3 75 ± 38 vs 303 ± 119 µg/dL, FBAL 13.7 ± 2.5 vs 19.7 ± 2.0 µg/mL, FA 204.0 ± 91.6 vs 395.9 ± 12.6 ng/mL, mean ± standard deviation, all p < 0.05). After seven cycles, partial response was confirmed. CONCLUSION: HD on day 2 instead of 3 may prevent hyperammonemia in 5FU/LV therapy.

Posttranslational modification of dinitrogenase reductase in Rhodospirillum rubrum treated with fluoroacetate.

Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.

Concentration-dependent changes to diffusion and chemical shift of internal standard molecules in aqueous and micellar solutions.

Sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) is the most widely accepted internal standard for protein NMR studies in aqueous conditions. Since its introduction as a reference standard, however, concerns have been raised surrounding its propensity to interact with biological molecules through electrostatic and hydrophobic interactions. While DSS has been shown to interact with certain proteins, membrane protein studies by solution-state NMR require use of membrane mimetics such as detergent micelles and, to date, no study has explicitly examined the potential for interaction between membrane mimetics and DSS. Consistent with its amphipathic character, we show DSS to self-associate at elevated concentrations using pulsed field gradient-based diffusion NMR measurements. More critically, DSS diffusion is significantly attenuated in the presence of either like-charged sodium dodecyl sulfate or zwitterionic dodecylphosphocholine micelles, the two most commonly used detergent-based membrane mimetic systems used in solution-state NMR. Binding to oppositely charged dodecyltrimethylammonium bromide micelles is also highly favourable. DSS-micelle interactions are accompanied by a systematic, concentration- and binding propensity-dependent change in the chemical shift of the DSS reference signal by up to 60 ppb. The alternative reference compound 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) exhibits highly similar behaviour, with reversal of the relative magnitude of chemical shift perturbation and proportion bound in comparison to DSS. Both DSS and DSA, thus, interact with micelles, and self-assemble at high concentration. Chemical shift perturbation of and modulation of micellar properties by these molecules has clear implications for their use as reference standards.

Biodegradation of mono-, di- and trifluoroacetate by microbial cultures with different origins.

This work focused on the biodegradation of three structurally related fluoroacetates (FAs), mono- (MFA), di- (DFA) and trifluoroacetate (TFA), using as microbial inocula samples collected from a site with a long history of industrial contamination and activated sludge obtained from a municipal wastewater treatment plant. Biodegradation experiments were carried out under different modes of substrate supplementation, which included (i) FAs fed as sole carbon sources; (ii) FAs (only for DFA and TFA) fed in co-metabolism with sodium acetate; and (iii) mixtures of MFA with DFA or TFA. Biodegradation of the target compounds was assessed through fluoride ion release. Defluorination was obtained in the cultures fed with MFA, while DFA and TFA were recalcitrant in all tested conditions. When present in mixture, DFA was shown to inhibit biodegradation of MFA, while TFA had no effect. A total of 13 bacterial isolates obtained from MFA degrading cultures were found to degrade 20mgL-1 of this compound, as single strains, when supplemented as a sole carbon source. Sequencing of the 16S rRNA gene indicated that among these degrading bacteria only Delftia acidovorans had been previously reported to be able to degrade MFA. This work shows that, despite their similar chemical structures, biodegradation of the three tested FAs is very distinct and draws attention to the unknown impacts that the accumulation of DFA and TFA may have in the environment as a result of their high recalcitrance.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

Protection against Amorimia septentrionalis poisoning in goats by the continuous administration of sodium monofluoroacetate-degrading bacteria.

The intraruminal inoculation of sodium monofluoroacetate (MFA)-degrading bacteria has been proposed as a method to prevent poisoning by MFA-containing plants. In previous experiments, MFA-degrading bacteria were inoculated intraruminally before or concurrent with plant challenge, with both strategies conferring partial protection to poisoning. To evaluate the protection to Amorimia septentrionalis poisoning provided by the continuous inoculation of MFA-degrading bacteria isolated from plants and soils, 18 goats were divided into three experimental groups of six animals each: Group 1 goats received daily doses of a mixture of Paenibacillus sp. and Cupriavidus sp., and Group 2 goats received a mixture of Ralstonia sp. and Burkholderia sp., for 40 days, while Group 3 goats were not inoculated. Ten days after initiation of bacterial inoculation in Groups 1 and 2, all goats were challenged daily with 5 g/kg body weight of green leaves from A. septentrionalis. Four goats from Group 1 consumed the leaves throughout the 30-day consumption period and showed clinical signs such as transient tachycardia and engorgement of the jugular. The two remaining animals from Group 1 showed obvious signs of intoxication, and plant administration was suspended on days 17 and 19. The goats in Group 2 consumed the leaves throughout the 30-day study without showing signs of poisoning. The goats from Group 3 (control) manifested severe clinical signs of poisoning between the 3rd and 10th days following the start of the A. septentrionalis challenge. Under the conditions of this experiment continuous intraruminal administration of Ralstonia sp. and Burkholderia sp. provided complete protection to poisoning by A. septentrionalis in goats, while continuous intraruminal administration of Paenibacillus sp. and Cupriavidus sp. provided partial protection.

Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

Fluoroacetate biosynthesis from the marine-derived bacterium Streptomyces xinghaiensis NRRL B-24674.

Genome sequencing identified a fluorinase gene in the marine bacterium Streptomyces xinghaiensis NRRL B-24674. Fermentation of the organism with inorganic fluoride (2 mM) demonstrated that the organism could biosynthesise fluoroacetate and that fluoroacetate production is sea-salt dependent. This is the first fluorometabolite producing microorganism identified from the marine environment.

Natural and engineered biosynthesis of fluorinated natural products.

Both natural products and synthetic organofluorines play important roles in the discovery and design of pharmaceuticals. The combination of these two classes of molecules has the potential to be useful in the ongoing search for new bioactive compounds but our ability to produce site-selectively fluorinated natural products remains limited by challenges in compatibility between their high structural complexity and current methods for fluorination. Living systems provide an alternative route to chemical fluorination and could enable the production of organofluorine natural products through synthetic biology approaches. While the identification of biogenic organofluorines has been limited, the study of the native organisms and enzymes that utilize these compounds can help to guide efforts to engineer the incorporation of this unusual element into complex pharmacologically active natural products. This review covers recent advances in understanding both natural and engineered production of organofluorine natural products.

Evaluation of 2-[¹8F]fluoroacetate kinetics in rodent models of cerebral hypoxia-ischemia.

Glia account for 90% of human brain cells and have a significant role in brain homeostasis. Thus, specific in vivo imaging markers of glial metabolism are potentially valuable. In the brain, 2-fluoroacetate is selectively taken up by glial cells and becomes metabolically trapped in the tricarboxylic acid cycle. Recent work in rodent brain injury models demonstrated elevated lesion uptake of 2-[(18)F]fluoroacetate ([(18)F]FACE), suggesting possible use for specifically imaging glial metabolism. To assess this hypothesis, we evaluated [(18)F]FACE kinetics in rodent models of cerebral hypoxia-ischemia at 3 and 24 hours post insult. Lesion uptake was significantly higher at 30 minutes post injection (P<0.05). An image-based method for input function estimation using cardiac blood was validated. Analysis of whole blood showed no significant metabolites and plasma activity concentrations of ∼50% that of whole blood. Kinetic models describing [(18)F]FACE uptake were developed and quantitatively compared. Elevated [(18)F]FACE uptake was found to be driven primarily by K1/k2 rather than k3, but changes in the latter were detectable. The two-tissue irreversible uptake model (2T3k) was found to be necessary and sufficient for modeling [(18)F]FACE uptake. We conclude that kinetic modeling of [(18)F]FACE uptake represents a potentially useful tool for interrogation of glial metabolism.

Degradation rate of sodium fluoroacetate in three New Zealand soils.

The degradation rate of sodium fluoroacetate (SFA) was assessed in a laboratory microcosm study incorporating 3 New Zealand soil types under different temperature (5 °C, 10 °C, or 20 °C) and soil moisture (35% or 60% water holding capacity) conditions using guideline 307 from the Organisation for Economic Co-operation and Development. A combination of nonlabeled and radiolabeled (14) C-SFA was added to soil microcosms, with sampling and analysis protocols for soil, soil extracts, and evolved CO(2) established using liquid scintillation counting and liquid chromatography-mass spectrometry. Degradation products of SFA and their rates of formation were similar in the 3 soil types. The major degradation pathway for SFA was through microbial degradation to the hydroxyl metabolite, hydroxyacetic acid, and microbial mineralization to CO(2), which constituted the major transformation product. Temperature, rather than soil type or moisture content, was the dominant factor affecting the rate of degradation. Soil treatments incubated at 20 °C displayed a more rapid loss of (14)C-SFA residues than lower temperature treatments. The transformation half-life (DT50) of SFA in the 3 soils increased with decreasing temperature, varying from 6 d to 8 d at 20 °C, 10 d to 21 d at 10 °C, and 22 d to 43 d at 5 °C.