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.     

Insights into fluorometabolite biosynthesis in Streptomyces cattleya DSM46488 through genome sequence and knockout mutants

Streptomyces cattleya DSM 46488 is unusual in its ability to biosynthesise fluorine containing natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S-adenosyl-l-methionine:fluoride adenosyltransferase (fluorinase, SCATT_41540), 5′-fluoro-5′-deoxyadenosine phosphorylase (SCATT_41550), fluoroacetyl-CoA thioesterase (SCATT_41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT_20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT_p11780) confirm that they are essential for fluorometabolite production. Notably gene disruption of the transaldolase (SCATT_p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesise 4-fluorothreonine, revealing a branchpoint role for the PLP-transaldolase.     

Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme

Fluorinated compounds are finding increasing uses in several applications. They are employed in almost all areas of modern society. These compounds are all produced by chemical synthesis and their abundance highly contrasts with fluorinated molecules of natural origin. To date, only some plants and a handful of actinomycetes species are known to produce a small number of fluorinated compounds that include fluoroacetate (FA), some ω-fluorinated fatty acids, nucleocidin, 4-fluorothreonine (4-FT), and the more recently identified (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid. This largely differs from other naturally produced halogenated compounds, which totals more than 5000. The mechanisms underlying biological fluorination have been uncovered after discovering the first actinomycete species, Streptomyces cattleya, that is capable of producing FA and 4-FT, and a fluorinase has been identified as the enzyme responsible for the formation of the C–F bond. The discovery of this enzyme has opened new perspectives for the biotechnological production of fluorinated compounds and many advancements have been achieved in its application mainly as a biocatalyst for the synthesis of [¹⁸F]-labeled radiotracers for medical imaging. Natural fluorinated compounds may also be derived from abiogenic sources, such as volcanoes and rocks, though their concentrations and production mechanisms are not well known. This review provides an outlook of what is currently known about fluorinated compounds with natural origin. The paucity of these compounds and the biological mechanisms responsible for their production are addressed. Due to its relevance, special emphasis is given to the discovery, characterization and biotechnological potential of the unique fluorinase enzyme.     

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.     

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.     

Genetically Modified Ruminal Bacteria Protect Sheep from Fluoroacetate Poisoning

Four strains of Butyrivibrio fibrisolvens, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10⁶ to 10⁷ cells ml⁻¹ in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.     

Stereochemical studies on a plasmid-coded fluoroacetate halidohydrolase

The stereochemical course of action of haloacetate halidohydrolase H-1 from Pseudomonas sp., strain A, which catalyzes the dehalogenation of fluoroacetate to glycolate, has been determined by enzymatic analysis of products from incubations with both enantiomers of 20-fluoropropionate, and by ¹H NMR analysis of the ester of (?)-α-methoxy-α-(trifluoromethyl)phenylacetic acid with phenacyl [2-²H₁]glycolate derived from the product of incubation with the (S)-monodeuterofluoroacetate. The results support a direct displacement mechanism for this enzyme, since they indicate that the reaction is catalyzed with inversion of configuration.     

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.     

Fluorocitrate inhibition of aconitase. Reversibility of the inactivation

Fluoride ion is released nearly stoichiometrically when (?)-erythro-fluorocitrate is incubated with aconitase. The release of F^? parallels the loss in activity and could arise from direct displacement of F^? by a base on the enzyme or from dehydration to fluoro-cis-aconitate and attack of an enzymic base to release F^?. Aconitase inactivated by ¹⁴C-fluorocitrate does not retain radioactivity when passed through G-50 Sephadex or precipitated by ammonium sulfate. Fullenzymicactivity can be regained after either of these treatments by activation by cysteine and ferrous salts. These data are consistent with the report of fluorocitrate being a competitive (and non-competitive) inhibitor of aconitase (Villafranca, J.J. (1972) Intra-Science Chem. Rept. 6 (4), 1–11) which rapidly inactivates the enzyme. This inactivated enzyme may be a very labile covalent complex, a very tight complex between enzyme and fluoro-cis-aconitate or a tight complex between a defluorinated deravitive of fluorocitrate.In the course of Peters (1957) extensive work on the toxic effects of fluoroacetate, he determined that fluoroacetate was metabolically converted to fluorocitrate. This finding and the fact that citrate levels rise soon after ingestion of fluoroacetate led to the suggestion that fluorocitrate inactivates aconitase (E.C. 4.2.1.2).Recently, conflicting reports concerning the site of inactivation in mitochondria by the inhibiting isomer of fluorocitrate, (?)-erythro-fluorocitrate (1R:2R, 1-fluoro-2-hydroxy-1,2,3-propanetricarboxylate) have appeared (Eanes et al. 1972; Brand et al, 1973). Eanes et al. (1972) contends that the tricarboxylate carrier is the site of inhibition, while Brand et al. (1973) has compelling evidence that aconitase is the site of inhibition. This controversy is a matter of intrepretation of the results and a greater knowledge of the inactivation of aconitase by fluorocitrate may be useful in these interpretations. The results reported herein are concerned with the mechanism of inactivation of purified mitochondrial aconitase by fluorocitrate and demonstrate that this reaction is readily reversible.     

Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr⁴², His⁷⁶, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg¹²⁰ located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.     

Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya

In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.     

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV–Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (k_cat/K_m) for indole was measured as 6.14 ± 0.10 min^− 1 mM^− 1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.     

Methionine inhibition of thienamycin formation

Summary Methionine interference in the formation of thienamycin byStreptomyces cattleya is due, to a major extent, to inhibition of enzyme activity.     

Experiments on the fluoroacetate metabolism of Dichapetalum cymosum (Gifblaar)

It was shown that application of fluoroacetate to leaf disks of Dichapetalum cymosum (Gifblaar) did not lead to an inhibition of oxygen uptake or accumulation of citrate, in contrast to the 'control plant' Parimarium capense which lacks fluoroacetate. The addition of fluorocitrate did, however, inhibit the oxygen uptake of both plants and caused an accumulation of citrate. From the results it was deduced that either citrate synthetase or acetate thiokinase from D. cymosum had different affinities for the fluorinated derivative and the 'normal' metabolite. The addition of fluoropyruvate to leaf disks caused a decrease in oxygen uptake and no change in the citrate concentration. From this it was deduced that fluoropyruvate inhibited pyruvate oxidase in both plants. It was concluded that the tolerance of D. cymosum to such high concentrations of fluoroacetate may be ascribed to the fact that the 'lethal synthesis' of fluorocitrate does not take place in the plant most probably because citrate synthetase has different affinities for fluoroacetylcoenzyme A and acetylcoenzyme A.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

Fluoroacetate toxicity inThiobacillus neapolitanus and its relevance to the problem of obligate chemoautotrophy

Summary Fluoroacetate was extremely toxic toThiobacillus neapolitanus strainC, retarding growth even at 10^-9 m. Inhibition could be relieved by relatively high concentrations of acetate or propionate. Inhibited cultures eventually recovered from inhibition and grew in the presence of fluoroacetate over the concentration range 10^-9 to 10^-5 m. The recovery represented a recommencement of division of the total population, as it was shown that 60–100% of the organisms inoculated on to agar containing as much as 10^-3 m fluoroacetate formed colonies after lags as long as 37 days. Even longer lags occurred with more fluoroacetate, but fewer organisms survived. Fluoroacetate appeared specifically to inhibit the Krebs' cycle through fluorocitrate synthesis; this confirmed that the cycle is essential to the autotrophic metabolism.Fluoroacetate-resistant variants occurred spontaneously at a frequency of about 2 per million organisms. These grew at normal exponential rates even in the presence of 10^-2 m fluoroacetate. They appeared to differ from the wild type organism only in lacking acetyl coenzyme A synthetase and possibly having decreased permeability to acetate. The origin of acetyl coenzyme A for biosynthesis in these mutants, and the significance of the lack of heterotrophic enzymes from an obligate autotroph, are discussed.     

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.     

The effect of sodium monofluoroacetate on plasma testosterone concentration in Tiliqua rugosa (gray)

• 1.1. Administration of multiple or single doses of sodium fluoroacetate (1080) to male Tiliqua rugosa caused a decrease in plasma testosterone concentration.
• 2.2. A single dose of 100 or 250 mg 1080 kg⁻¹ body weight decreased plasma testosterone by 52%. However, 25 mg kg⁻¹ had little apparent effect on testosterone levels. When lizards were given the multiple dose equivalent of these doses over 12 days at 3 day intervals, the effect was much less dramatic with plasma testosterone concentration steadily declining over 15 days for the two higher doses.
• 3.3. There was a suggestion of degeneration of seminiferous tubules in some individuals.

     

Electron transport to nitrogenase in Rhodospirillum rubrum: the role of NAD(P)H as electron donor and the effect of fluoroacetate on nitrogenase activity

The role of the reactions of the TCA cycle in the generation of reductant for nitrogenase in Rhodospirillum rubrum has been investigated. Addition of fluoroacetate inhibited nitrogenase activity almost completely when pyruvate or endogenous sources were used as electron donors, whereas the inhibition was incomplete when malate, succinate or fumarate were used. Addition of NAD(P)H to cells supported nitrogenase activity, both with and without prior addition of fluoroacetate. We suggest that the role of the TCA cycle in nitrogen fixation in R. rubrum is to generate reduced pyridine nucleotides which are oxidized by the components of the electron transport pathway to nitrogenase.     

In vivo regulation of glutamine synthetase by ammonium in the cyanobacterium Anabaena L-31

In cell-free preparations of NH₄⁺-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N₂-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH₄⁺-mediated repression. Glutamine synthetase activity in both N₂-grown and NH₄⁺-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N₂-grown cultures, a drastic decrease in the enzyme activity by exogenous NH₄⁺ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has K_m values for NH₄⁺, glutamate Mg²⁺, and ATP similar to those observed for the enzyme from N₂- and NH₄⁺-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH₄⁺-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N₂-fixing cultures of Anabaena.