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At least 19 recordsLinked to original sources
PubMed1997

Detection and molecular analysis of plant- and insect-associated bacteria harboring aconitate isomerase involved in biosynthesis of trans-aconitic acid as antifeedant in brown planthoppers.

The activity of aconitate isomerase, which is involved in the biosynthesis of trans-aconitic acid as antifeedant in brown planthoppers, was detected in Pseudomonas fluorescens LRB3W1 and Pseudomonas putida MAFF301685 but not in Pseudomonas putida MAFF301684. The enzyme activity was induced in the presence of trans-aconitate, and therefore bacteria showing the enzyme activity were easily detected by their ability to grow on the minimal medium containing trans-aconitate as the sole carbon source (ACO agar medium). Experiments on growth of plant- or insect-associated bacteria on ACO agar medium showed that most of the Gram-negative bacteria displayed the aconitate isomerase activity unlike most of the Gram-positive bacteria isolated mainly from insects. Mini-Tn5 transposon derivatives of P. fluorescens LRB3W1 lacking completely or partially their ability to grow on ACO agar medium were obtained. Southern blot analysis with a mini-Tn5 DNA probe definitely showed that the genes responsible for the biosynthesis of aconitate isomerase present on chromosomal DNA. Thus, it was suggested that genes for aconitate isomerase biosynthesis are commonly present in Gram-negative plant- or insect-associated bacteria, and also the DNA fragments including the genes were detected in P. fluorescens LRB3W1.

Aconitic Acid↗
PubMed1994

Ability of Acidaminococcus fermentans to oxidize trans-aconitate and decrease the accumulation of tricarballylate, a toxic end product of ruminal fermentation.

Mixed ruminal bacteria convert trans-aconitate to tricarballylate, a tricarboxylic acid which chelates blood divalent cations and decreases their availability (J. B. Russell and P. J. Van Soest, Appl. Environ. Microbiol. 47:155-159, 1984). Decreases in blood magnesium in turn cause a potentially fatal disease known as grass tetany. trans-Aconitate was stoichiometrically reduced to tricarballylate by Selenomonas ruminantium, a common ruminal bacterium in grass-fed ruminants (J. B. Russell, Appl. Environ. Microbiol. 49:120-126, 1985). When mixed ruminal bacteria were enriched with trans-aconitate, a trans-aconitate-oxidizing bacterium was also isolated (G. M. Cook, F. A. Rainey, G. Chen, E. Stackebrandt, and J. B. Russell, Int. J. Syst. Bacteriol. 44:576-578, 1994). The trans-aconitate-oxidizing bacterium was identified as Acidaminococcus fermentans, and it converted trans-aconitate to acetate, a nontoxic end product of ruminal fermentation. When S. ruminantium and A. fermentans were cocultured with trans-aconitate and glucose, tricarballylate never accumulated and all the trans-aconitate was converted to acetate. Continuous-culture studies (dilution rate, 0.1 h-1) likewise indicated that A. fermentans could outcompete S. ruminantium for trans-aconitate. When mixed ruminal bacteria were incubated in vitro with 10 mM trans-aconitate for 24 h, 45% of the trans-aconitate was converted to tricarballylate. Tricarballylate production decreased 50% if even small amounts of A. fermentans were added to the incubation mixes (0.01 mg of protein per mg of mixed bacterial protein). When A. fermentans (2 g of bacterial protein) was added directly to the rumen, the subsequent conversion of trans-aconitate to tricarballylate decreased 50%, but this effect did not persist for more than 18 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitic Acid↗
PubMed1969

The inhibition by fluorocitrate of rat liver mitochondrial and extramitochondrial aconitate hydratase.

1. The effects of synthetic fluorocitrate were studied on: (a) the oxidation of citrate and cis-aconitate by rat liver mitochondria; (b) the activity of the aconitate hydratase found in the liver cell sap; (c) the activity of the aconitate hydratase solubilized from liver mitochondria. 2. Fluorocitrate was found to be a potent inhibitor of oxidation of citrate but only a weak inhibitor of oxidation of cis-aconitate: 6.7mum-fluorocitrate (containing 4% of the inhibitory isomer) caused 94% inhibition of the oxidation of citrate (2mm) whereas 1.0mm-fluorocitrate was necessary to provoke the same inhibition when cis-aconitate (2mm) was the substrate. The degree of inhibition varied in relation to the respiratory state of mitochondria when fluorocitrate was added. The inhibition could be partially reversed by cis-aconitate. 3. The aconitate hydratase extracted from the mitochondria was much less inhibited by fluorocitrate than was the mitochondria-bound enzyme, and the aconitate hydratase found in the cell sap was even less sensitive. 0.3mm-Fluorocitrate was required to cause 50% inhibition of the reaction citrate-->cis-aconitate, catalysed by the aconitate hydratase extracted from the mitochondria, and 1.2m-fluorocitrate for the extramitochondrial enzyme. For both enzymes the reaction citrate-->cis-aconitate was 2-3 times more sensitive to fluorocitrate than was the reaction isocitrate-->cis-aconitate. The inhibition was of the competitive type for both reactions.

Animals↗
PubMed2001

Identification of the gene and characterization of the activity of the trans-aconitate methyltransferase from Saccharomyces cerevisiae.

We have identified the yeast open reading frame YER175c as the gene encoding the trans-aconitate methyltransferase of Saccharomyces cerevisiae. Extracts of a yeast strain with a disrupted YER175c gene demonstrate a complete loss of activity toward the methyl-accepting substrates trans-aconitate, cis-aconitate, DL-isocitrate, and citrate. Reintroduction of the YER175c gene on a plasmid results in an overexpression of the activity toward each of these methyl-accepting substrates. We now designate this gene TMT1 for trans-aconitate methyltransferase. We examined the methyl-accepting substrate specificity of this enzyme in extracts from overproducing cells. We found that trans-aconitate was the best substrate with a Km of 0.66 mM. Other substrates were recognized much more poorly, including cis-aconitate with a Km of 74 mM and the decarboxylation product itaconate with a Km of 44 mM. The ratio of the maximal velocity to the Km of these substrates was only 0.24% and 0.9% that of trans-aconitate; for other substrates including citrate and other tricarboxylate and dicarboxylate derivatives, this ratio ranged from 0.0003% to 0.062% that of trans-aconitate. We then asked if any of these compounds were present endogenously in yeast extracts. We were able to identify trans-aconitate 5-methyl ester as well as additional unidentified radiolabeled products when S-adenosyl-L-[methyl-3H]methionine was mixed with TMT1+ extracts (but not with tmt1- extracts), suggesting that there may be additional substrates for this enzyme. We showed that the product 5-methyl ester of trans-aconitate is not readily metabolized in yeast extracts. Finally, we demonstrated that the activity of the yeast trans-aconitate methyltransferase is localized in the cytosol and increases markedly as cells undergo the metabolic transition at the diauxic shift.

Amino Acid Sequence↗
PubMed2003

[Study on the combination principles about aconite roots by electrospray ionization mass spectrometry].

AIM: To explore the combination principle about aconite roots from the chemical point of view. METHODS: Analyzing and comparing the nordeterpenoid alkaloids in the crude aconite roots, the decoction of crude aconite roots and aconite roots with some other Chinese traditional herbs by electrospray ionization trap tandem mass spectrometry. Detecting the pH value of the decoction of crude aconite roots and of some other Chinese traditional herbs; studying the influence of acidity to the hydrolysis of standard sample of mesaconitine. RESULTS: Most of diester-diterpenoid alkaloids in the decoction of aconite roots and the decoction of aconite roots with liquorice roots are hydrolyzed, and the hydrolysis of alkaloids are inhibited in the decoction of aconite roots with Pinellia tuber or Schisandra. The acidity of the decoction of aconite roots or liquorice is lower than the decoction of Pinellia tuber or Schisandra; standard sample of mesaconitine can not be hydrolyzed in acid water. CONCLUSION: Acidity is a key factor to influence the hydrolysis of diester-diterpenoid alkaloids, and the hydrolysis of alkaloids are inhibited in high acidity, which lead to the high toxicity of the decoction containing aconite roots.

Aconitine↗
PubMed1988

Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells.

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.

Aconitate Hydratase↗
PubMed2003

Exploring the ester-exchange reactions of diester-diterpenoid alkaloids in the aconite decoction process by electrospray ionization tandem mass spectrometry.

The chemical components in the decoctions of Chinese herbal medicines are not always the same as those in the crude herbs because of the insolubility or instability of some compounds. In this work electrospray ionization tandem mass spectrometry was used to explore the ester-exchange reactions for aconitine-type diester-diterpenoid alkaloids occurring during the process of decocting aconite root. The aconitines were screened in a diverse range of samples, including crude aconite, decoction of crude aconite, residues from decoction of crude aconite, prepared aconite, decoction of prepared aconite, decoction of prepared aconite with added palmitic acid, and decoction of a mixture of mesaconitine and hypaconitine standards with liquorice root. It was found that diester-diterpenoid aconitines were converted into lipo-alkaloids as well as monoester alkaloids by the decoction of aconite.

Aconitum↗
PubMed1971

Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase.

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1-0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01-0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the K(i) values calculated were 1x10(-6)m for the mitochondrial and 2.5x10(-6)m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate-->cis-aconitate more than that of the reaction isocitrate-->cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.

Animals↗
PubMed1999

A novel methyltransferase catalyzes the methyl esterification of trans-aconitate in Escherichia coli.

We have identified a new type of S-adenosyl-L-methionine-dependent methyltransferase in the cytosol of Escherichia coli that is expressed in early stationary phase under the control of the RpoS sigma factor. This enzyme catalyzes the monomethyl esterification of trans-aconitate at high affinity (Km = 0.32 mM) and cis-aconitate, isocitrate, and citrate at lower velocities and affinities. We have purified the enzyme to homogeneity by gel-filtration, anion-exchange, and hydrophobic chromatography. The N-terminal amino acid sequence was found to match that expected for the o252 open reading frame at 34.57 min on the E. coli genomic sequence whose deduced amino acid sequence contains the signature sequence motifs of the major class of S-adenosyl-L-methionine-dependent methyltransferases. Overexpression of the o252 gene resulted in an overexpression of the methyltransferase activity, and we have now designated it tam for trans-aconitate methyltransferase. We have generated a knock-out strain of E. coli lacking this activity, and we find that its growth and stationary phase survival are similar to that of the parent strain. We demonstrate the endogenous formation of trans-aconitate methyl ester in extracts of wild type but not tam- mutant cells indicating that trans-aconitate is present in E. coli. Since trans-aconitate does not appear to be a metabolic intermediate in these cells but forms spontaneously from the key citric acid cycle intermediate cis-aconitate, we suggest that its methylation may limit its potential interference in normal metabolic pathways. We have detected trans-aconitate methyltransferase activity in extracts of the yeast Saccharomyces cerevisiae, whereas no activity has been found in extracts of Caenorhabditis elegans or mouse brain.

Amino Acid Sequence↗
PubMed2004

3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase.

The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may attenuate trans-aconitate inhibition of aconitase and other enzymes of the cycle. Although trans-aconitate is a minor endogenous substrate of the Tmt1 enzyme in extracts of S. cerevisiae, the major endogenous substrate has yet to be identified. We show here that a trimethylsilylated derivative of the major methylated endogenous product of Tmt1 in yeast extracts has an identical gas chromatography retention time and an identical electron impact mass spectrum as one of the two possible monomethyl ester derivatives of (2R,3S)-3-isopropylmalate. (2R,3S)-3-Isopropylmalate is an intermediate of the leucine biosynthetic pathway that shares similar intermediates and reaction chemistry with the portion of the citric acid cycle from oxaloacetate to alpha-ketoglutarate via cis-aconitate. The Tmt1 methyltransferase recognizes (2R,3S)-3-isopropylmalate with similar kinetics as it does trans-aconitate, with respective K(m) values of 127 and 53 microM and V(max) values of 59 and 70 nmol min(-1) mg(-1) of protein in a Tmt1-overexpressed yeast extract. However, we found that isopropylfumarate, the direct homologue of trans-aconitate in the leucine biosynthetic pathway, was at best a very poor substrate for the Tmt1 yeast enzyme. Similarly, the direct homologue of 3-isopropylmalate in the citric acid cycle, isocitrate, is also a very poor substrate. This apparent change in specificity between the intermediates of these two pathways can be understood in terms of the binding of these substrates to the active site. These results suggest that the Tmt1 methyltransferase may work in two different pathways in two different ways: for detoxification in the citric acid cycle and for a possibly novel biosynthetic branch reaction of the leucine biosynthetic pathway.

Aconitic Acid↗
PubMed1963

MICROBIOLOGICAL DISSIMILATION OF TRICARBALLYLATE AND TRANS-ACONITATE.

Altekar, W. W. (National Chemical Laboratory, Poona, India) and M. R. Raghavendra Rao. Microbiological dissimilation of tricarballylate and trans-aconitate. J. Bacteriol. 85:604-613. 1963.-Two fluorescent pseudomonads capable of metabolizing tricarballylate and trans-aconitate were isolated by the soil-enrichment culture technique. These and some other species of bacteria were tested for their ability to utilize for growth the salts of many di- and tricarboxylic acids. Alloisocitrate and mesaconate were not utilized by any of the ten strains tested; only two strains grew on tricarballylate and itaconate. trans-Aconitate was utilized by many strains which had not been previously exposed to this compound. The resting cells of two strains could adapt to oxidize two acids (tricarballylate and trans-aconitate), and this induction was chloramphenicol-sensitive. The tricarballylate-grown cells were simultaneously induced to oxidize trans-aconitate and other tricarboxylates, whereas the trans-aconitate-grown cells were not induced to oxidize tricarballylate, and their subsequent induction was inhibited by chloramphenicol. This trans-aconitate or tricarballylate. But tricarballylate dehydrogenase was present only in tricarballylate-grown cells. The cell-free extracts of the two organisms contained the enzymes of the Krebs cycle and isocitritase. These enzymes are most probably operative during growth on and oxidation of these two acids as sole carbon sources.

Aconitic Acid↗
PubMed1994

Crystal structures of aconitase with trans-aconitate and nitrocitrate bound.

Crystal structures of mitochondrial aconitase with the inhibitors trans-aconitate and nitrocitrate bound to the [4Fe-4S] cluster have been solved and refined at 2.05 A resolution with R-factors of 0.168 and 0.172, respectively. Crystallization of aconitase with the substrates citrate and cis-aconitate has not been possible because the enzyme turns over and selects enzyme with isocitrate bound into the crystal lattice. Therefore we have analyzed crystal structures of the enzyme complexed with inhibitor analogs of these two substrates. The structure with nitrocitrate bound provides a model for citrate binding. The structure with trans-aconitate bound provides a model for cis-aconitate binding in two ways: Fe4 of the [4Fe-4S] cluster is five-coordinate and the carbon at the C beta position is trigonal. These results allow the model for the reaction mechanism to be extended to all three natural substrates of aconitase. The results support a model in which citrate and isocitrate form similar chelate structures related by 180 degrees rotation about the C alpha-C beta bond while the intermediate cis-aconitate binds in either of two ways (citrate mode or isocitrate mode). In both inhibitor complexes a H2O molecule is also bound to Fe4. In the structure with nitrocitrate bound, partial occupancy of sulfate in the active site is observed accompanied by hydroxyl binding to Fe4. Comparison of the structures with isocitrate, trans-aconitate, nitrocitrate and sulfate bound reveals preferred orientations for the three types of oxygens ligated to Fe4 (carboxyl, hydroxyl and H2O) supporting the proposed roles for His101, Asp165 and His167 in the catalytic mechanism.

Aconitate Hydratase↗
PubMed1990

Determination of aconitate isomerase in plants.

We have developed a method for measuring aconitate isomerase (EC 5.3.3.7) in plants which depends on the release of tritium from labeled trans-aconitate. The released tritium is separated from labeled aconitate by passage through a column of strong anion-exchange resin. This method is more sensitive, simpler, and more specific than previous methods, especially with crude extracts. The validity of the method was demonstrated by a comparison of the quantity of tritium released with the amount of cis-aconitate isomerized and this comparison demonstrated an isotope effect. Aconitase did not interfere. The method was tested by measuring aconitate isomerase in crude extracts of several higher plant species and tissues and these analyses showed that wheat and corn have more aconitate isomerase than the other species tested.

Aconitate Hydratase↗
PubMed2001

Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases.

The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries.

Aconitic Acid↗
PubMed1985

Enrichment and Isolation of Rumen Bacteria That Reduce trans- Aconitic Acid to Tricarballylic Acid.

Bacteria from the bovine rumen capable of reducing trans-aconitate to tricarballylate were enriched in an anaerobic chemostat containing rumen fluid medium and aconitate. After 9 days at a dilution rate of 0.07 h, the medium was diluted and plated in an anaerobic glove box. Three types of isolates were obtained from the plates (a crescent-shaped organism, a pleomorphic rod, and a spiral-shaped organism), and all three produced tricarballylate in batch cultures that contained glucose and trans-aconitate. In glucose-limited chemostats (0.10 h), trans-aconitate reduction was associated with a decrease in the amount of reduced products formed from glucose. The crescent-shaped organism produced less propionate, the pleomorphic rod produced less ethanol, and the spiral made less succinate and possibly H(2). Aconitate reduction by the pleomorphic rod and the spiral organism was associated with a significant increase in cellular dry matter. Experiments with stock cultures of predominant rumen bacteria indicated that Selenomonas ruminantium, a species taxonomically similar to the crescent-shaped isolate, was an active reducer of trans-aconitate. Strains of Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Megasphaera elsdenii produced little if any tricarballylate. Wolinella succinogenes produced some tricarballylate. Based on its stability constant for magnesium (K(eq) = 115), tricarballylate could be a factor in the hypomagnesemia that leads to grass tetany.

Journal Article↗