Isolation of an Aldehyde Dehydrogenase Involved in the Oxidation of Fluoroacetaldehyde to Fluoroacetate in Streptomyces cattleya

Streptomyces cattleya is unusual in that it produces fluoroacetate and 4-fluorothreonine as secondary metabolites. We now report the isolation of an NAD⁺-dependent fluoroacetaldehyde dehydrogenase from S. cattleya that mediates the oxidation of fluoroacetaldehyde to fluoroacetate. This is the first enzyme to be identified that is directly involved in fluorometabolite biosynthesis. Production of the enzyme begins in late exponential growth and continues into the stationary phase. Measurement of kinetic parameters shows that the enzyme has a high affinity for fluoroacetaldehyde and glycoaldehyde, but not acetaldehyde.     

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.     

The identification of 5'-fluoro-5-deoxyinosine as a shunt product in cell free extracts of Streptomyces cattleya

5'-Fluoro-5'-deoxyinosine (5'-FDI) is identified as an adventitious side product that accumulates in cell free incubations of SAM and fluoride ion in Streptomyces cattleya. 5'-FDI was identified by a combination of isotopic labelling studies and co-synthesis studies as well as enzymatic degradation. Although it is an efficiently generated end product of the cell free incubations, 5'-FDI is not a biosynthetic intermediate and it does not accumulate as a fluorometabolite with fluoroacetate and 4-fluorothreonine in whole cell incubations of S. cattleya. Clearly the purine deaminase which converts 5'-fluoro-5'-deoxyadenosine (5'-FDA) to 5'-FDI in the cell free extract does not come into contact with 5'-FDA in whole cells, suggesting some level of compartmentalisation in cells of S. cattleya. The biotransformation of 5'-FDI from fluoride ion extends the range of organofluorine products, beyond biosynthetic intermediates, that can be generated by this system, for applications such as enzymatic labelling with fluorine-18 for positron emission tomography applications.     

The identification of (3R,4S)-5-fluoro-5-deoxy-D-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya

(3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.     

Temporal and fluoride control of secondary metabolism regulates cellular organofluorine biosynthesis

Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into small molecules of interest. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine-producing pathway, Streptomyces cattleya. Interestingly, we have discovered that the major mode of S. cattleya's resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host's metabolic machinery to discriminate between fluorinated and non-fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl-CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products.     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

Is fluoroacetate-specific defluorinase a glutathione S-transferase?

Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.     

Insulin-like effects of fluoroacetate on lipolysis and lipogenesis in adipose tissue.

Hormone-stimulated lipolysis in adipose tissue was inhibited by fluoroacetate and there was a concomitant decrease in both the basal and hormone-stimulated cyclic AMP levels. Adenylate cyclase (EC 4.6.1.1) activity in membrane preparations was inhibited by fluoroacetate. There was no influence of fluoroacetate on the low Km cyclic AMP phosphodiesterase (EC 3.1.4.17) activity. The rate of glucose conversion to fatty acids was increased when adipose tissue was incubated in the presence of fluoroacetate. The outputs of pyruvate and lactate into the incubation medium were decreased at this time, suggesting decreased tissue pyruvate levels and a site of activation of lipogenesis distal to pyruvate formation. Pyruvate dehydrogenase (EC 1.2.4.1) activity was increased twofold in adipose tissue incubated in the presence of fluoroacetate. This was attributed to a fluoroacetate-induced inhibition of pyruvate dehydrogenase kinase, the enzyme responsible for inactivating the pyruvate dehydrogenase complex. Glucose transport was increased to a small but significant degree by fluoroacetate. In addition, both the tissue content of citrate and its release into the incubation medium were increased, suggesting that fluoroacetate resulted in an inhibition of aconitase (EC 4.2.1.3). The tissue ATP content was unchanged. Because the antilipolytic and lipogenic effects of fluoroacetate parallel those of insulin, they may share a common mechanism.     

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Correlative biochemical and electron microscopic alterations were observed in chick embryo myoblasts in vitro after treatment with fluoroacetate. Fluoroacetate poisoning caused an increase of citrate and a decrease of ATP in the cultures. Cell respiration was only slightly impaired by fluoroacetate in the first 10 min but was inhibited to 30% one hour after exposure to the poison. Fluoroacetate did not affect oxidative phosphorylation. The evidence suggests that fluoroacetate was transformed in myoblasts into fluorocitrate which inhibited the mitochondrial-bound aconitate hydratase as in adult tissues. Ultrastructural changes in the majority of the fluoroacetate-treated cells were observed. Very few myoblasts appeared unaffected by the poison. Mitochondria were specifically altered. The early changes occurred in the mitochondrial matrix where the inhibited enzyme is known to be located and were followed by modifications in the configuration and structure of cristae. Exogenous fluorocitrate caused ultrastructural changes in the mitochondria similar to that provoked by fluoroacetate. The localization of the early change in the mitochondrial matrix and the evaluation of the structural modifications suggest a correlation between the biochemical lesion, i.e. the inhibition of aconitate hydratase, and the change revealed in the mitochondrial structure containing the inhibited enzyme.This work was supported by grants of the Consiglio Nazionale delle Ricerche to both InstitutesThe present study is dedicated to Prof. Otto Bucher on occasion of his 65th birthday     

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

Significance of sulfhydryl compounds in the manifestation of fluoroacetate toxicity to the rat, brush-tailed possum, woylie and western grey kangaroo

Levels of citrate in kidneys and livers of rats with normal glutathione levels increased 6.8 and 1.7-fold respectively 2 h after dosing with 1.5 mg of compound 1080 (= 95% sodium fluoroacetate) per kilogram body weight. In animals with liver glutathione levels 15% of normal, increases in plasma and liver citrate levels after dosing with fluoroacetate were significantly greater than those of control animals. Cysteamine and N-acetylcysteine, like glutathione, partially protected aconitate hydratase from fluorocitrate inhibition in rat liver preparations but were unable to replace glutathione as a substrate for the defluorination of fluoroacetate in vitro. N-Acetylcysteine did not diminish plasma citrate levels of glutathione-deficient rats dosed with fluoroacetate, while cysteamine inhibited the rate of in vivo defluorination in glutathione-deficient brush-tailed possums. It is suggested that non-physiological sulfhydryl compounds are ineffective antidotes to fluoroacetate intoxication in vivo. The in vivo defluorination patterns of four mammal species with differing sensitivities to fluoroacetate did not indicate a direct relationship between tolerance and rate of defluorination and it is also suggested that a high level of activity of the glutathione-S-transferase responsible for the defluorination of fluoroacetate is not the major mechanism for circumventing fluoroacetate toxicity in resistant mammals.     

Purification and properties of fluoroacetate dehalogenase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> DSM 8341

The degradation of fluoroacetate by microorganisms has been established for some time, although only a handful of dehalogenases capable of hydrolyzing the stable C–F bond have been studied. Pseudomonas fluorescens DSM 8341 was originally isolated from soil and readily degrades fluoroacetate, thus it was thought that its dehalogenase might have some desirable properties. The enzyme was purified from cell-free extracts and characterised: it is a monomer of 32,500 Da, with a pH optimum of 8 and is stable between pH 4 and 10; its activity is stimulated by some metal ions (Mg²⁺, Mn²⁺ and Fe³⁺), but inhibited by others (Hg²⁺, Ag²⁺). The enzyme is specific for fluoroacetate, and the K _m for this substrate (0.68 mM) is the lowest determined for enzymes of this type that have been investigated to date.     

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Chick embryo heart fragments in primary hanging-drop culture were treated with sodium fluoroacetate to induce inhibition of aconitate hydratase, a mitochondrial enzyme of the tricarboxylic acid cycle. The mitochondria were analyzed in the living myoblasts by phase-contrast time-lapse cinemicrography. The results were recorded in a 16 mm film. After 20–30 minutes contact of the cells with the inhibitor some mitochondria became thickened and swollen. The swelling was polymorphous, asynchronous and reversible; the same mitochondrion could swell and shrink many times. Some mitochondria seemed not to respond to fluoroacetate and remained rod-like. Mitochondria appeared the only cell components to be morphologically affected by fluoroacetate and the changes were specifically caused by the inhibitor. The type of mitochondrial swelling differed from the large-amplitude respiration-dependent swelling of the isolated mitochondria in vitro and from the configurational changes of isolated mitochondria associated with the respiratory states. The evidence pointed to a specific connection between the biochemical lesion caused by fluoroacetate and the configurational changes of the mitochondria. The mitochondrial swelling was to a large extent reversed by washing the cultures with Tyrode physiological saline solution and the reversal was further accentuated by incubation of the cultures in fresh nutrient medium.This work was supported by grants of the Consiglio Nazionale delle Richerce of Italy to both Institutes.     

Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis

Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined. Levels of GS in S. cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium. Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity. Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme. The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells. The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen. In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence.     

Studies on the Metabolism of Fluoroacetate in the Liver of a Rat

In a rat poisoned with sodium fluoroacetate no accumulation of citrate was found in the liver, while in that of a frog fluoroacetate adtninistration was found to cause a marked increase of citrate. In this paper, it was shown that even in a rat when it was treated with insulin before fluoroacetate administration, citrate accuillulation could be found in the liver. In the experiments in vitro using slices of a rat liver it was also shown that fluoroacetate could cause an accumulation of citrate and inhibited the oxidation of malate in the slices of a normal as well as an insulinized liver.     

Studies on the Abnormal Metabolism in Animals Poisoned with Sodium Fluoroacetate

The changes of metabolisms in the calf, dog and rat following administeration of fluoroacetate were studied. It was found that citrate is accumulated in the tissues, and increased amounts of acetone bodies are excreted in the urine of the animals which received fluoroacetate, and their urinary nitrogen also increases. All these abnormal accumulations and excretions of the rat after injection of fluoroacetate diminish within 24hrs., suggesting the decrease of the toxity or the excretion of the poison substance.

Significant amounts of organic fluorine compounds were detected in various tissues of fluoroacetate-treated calves.

The lethal dose limit of fluoroacetate for the calf could not be determined. As for the dog, 0.02 mg/kilo of fluoroacetate seemed to be fatal.     

Catalysis-linked inactivation of fluoroacetate dehalogenase by ammonia: a novel approach to probe the active-site environment

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp(105) of the enzyme acts as a nucleophile to attack the alpha-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His(272) [Liu, J.Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated concomitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp(105), into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for characterizing the active-site environment as well as for identifying the catalytic residue.     

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.     

Metabolism of fluoroacetate in the skink (Tiliqua rugosa) and the rat (Rattus norvegicus)

Administration of 100 mg sodium fluoroacetate (compound 1080) per kilogram body weight to T. rugosa resulted in a 3.4-fold increase in plasma citrate levels 48 h after dosing while administration of 3 mg sodium fluoroacetate per kilogram body weight to R. norvegicus produced a fivefold increase in plasma citrate levels within 4 h. Administration of 300 mg sodium fluoroacetate per kilogram body weight reduced the oxygen consumption of the skink by between 2.5 and 11% while in the rat, 2 mg sodium fluoroacetate per kilogram body weight reduced oxygen consumption by between 28 and 57%. Aconitate hydratase activity in extracts of liver acetone powders from T. rugosa was less inhibited by (-)erythrofluorocitrate (Ki: 0.065 mM) than that in extracts derived from R. norvegicus (Ki: 0.026 mM). The rate of defluorination of fluoroacetate in erythrocytes and in extracts of liver acetone powders of T. rugosa was 8- and 4.5-fold greater, respectively, than that found in similar preparations from R. norvegicus. A rapid rate of defluorination together with a low reliance on aerobic respiration favoured detoxification of fluoroacetate in T. rugosa rather than its conversion into fluorocitrate. Though defluorination in this species helped to minimize the immediate effects of fluoroacetate on aerobic respiration, it resulted in rapid depletion of liver glutathione levels.     

Detection and measurement of fluoroacetate in plant extracts by 19FNMR

Examination of extracts from seeds and foliage of several species known to contain fluoroacetate, using¹³F NMR spectroscopy, has shown the presence of the characteristic FCH₂-signal in most of them and enabled quantitative determination of their fluoroacetate content. No other fluorine-containing plant metabolites were detected; fluoroacetate was not detected in the extracts of several non-toxic species. The limit of detection is estimated to beca 4 μg/g.