Isolation of a Citrobacter species able to grow on malonate under strictly anaerobic conditions

An anaerobic enrichment from lake mud yielded a pure culture of a facultatively anaerobic bacterium able to grow on malonate under strictly anaerobic conditions. Strain 16mal1 was identified as a member of the family Enterobacteriaceae, and assigned to the genus Citrobacter on the basis of morphological, metabolic and biochemical characteristics. Malonate was fermented under strictly anaerobic (sulphide-reduced) conditions to acetate and CO2 concomitant with growth. A maximum growth rate of 1.88 generations h-1 (mu = 1.30 h-1) was measured. The dry weight yield of cells from malonate was estimated at 2.5 g mol-1. Yeast extract was required for growth on malonate: other additives, or a vitamin solution, could not replace this requirement. Other dicarboxylic acids were not degraded in the absence or presence of malonate. Malonate was degraded under anaerobic, but not aerobic conditions. Malonate-decarboxylating activity was inducible by malonate under both anaerobic and aerobic conditions, and was not expressed in glucose- or citrate-grown anaerobic cultures. Monensin had no effect on malonate degradation, while 2,4-dinitrophenol decreased the rate of malonate degradation. This, with the lack of a sodium requirement for anaerobic growth on malonate, suggested that ATP generation may not be mediated by a sodium-pumping mechanism.     

Protective effect of inhibitors of succinate dehydrogenase on wheat seedlings during osmotic stress

The effect of malonate and sedaxane, a compound with the fungicidal effect which act as succinate dehydrogenase inhibitors, on the resistance of etiolated wheat seedlings (Triticum aestivum L.) to osmotic stress caused by 12% PEG 6000 solution, was studied. The presowing treatment of seeds with 0.3 mM sedaxane solution significantly reduced the inhibitory effect of osmotic stress on seedling growth. The protective effect of 10 mM malonate was significant when it was added to the incubation medium of the roots; the effect of preseeding treatment with malonate was less significant. Unlike malonate, malate had no positive effect on seedling growth under osmotic stress. The activity of succinate dehydrogenase and the hydrogen peroxide content decreased in seedlings after the treatment of roots with malonate and sedaxane. Pretreatment with sedaxane and the addition of malonate to the incubation medium of roots prevented the accumulation of a lipid peroxidation product, malondialdehyde, which is caused by osmotic stress, and increased peroxidase activity. It was concluded that the stress-protective effect of sedaxane and malonate on wheat seedlings might be due to the inhibition of succinate dehydrogenase-dependent formation of reactive oxygen species and the prevention of oxidative cell damage.     

Inhibition of Nitric Oxide Synthase Activity Attenuates Striatal Malonate Lesions in Rats

Abstract: Mitochondrial inhibitors such as malonate are potent neurotoxins in vivo. Intrastriatal injections of malonate result in neuronal damage reminiscent of "excitotoxic" lesions produced by compounds that activate NMDA receptors. Although the mechanism of cell death produced by malonate is uncertain, overactivation of NMDA receptors may be involved; pretreatment of animals with NMDA antagonists provides neuroprotection against malonate lesions. NMDA receptor activation stimulates the enzyme nitric oxide (NO) synthase (NOS). Elevated tissue levels of NO may generate highly reactive intermediates that impair mitochondrial function. We hypothesized that NO may be a mediator of malonate toxicity. We investigated whether in vivo inhibition of NO production by the NOS inhibitor N ^ω-nitro- l -arginine (NLA) would attenuate lesions produced by intrastriatal injections of malonate. We found that systemic injections of 3 mg/kg of NLA significantly reduced the extent of histologic damage elicited by intrastriatal injections of 1.5 µmol of malonate in adult rats.     

Growth and synthesis of rubratoxin by Penicillium rubrum in a chemically defined medium fortified with organic acids and intermediates of the tricarboxylic acid cycle

A sterile glucose-mineral salts broth was fortified with equimolar concentrations (10-³ M) of various organic acids and intermediates in the tricarboxylic acid cycle. Appropriate media were neutralized with 2 N NaOH, inoculated with spore suspensions or mycelial pellets ofPenicillium rubrum and incubated quiescently for 14 days or with shaking for 5 days. Rubratoxins were recovered from culture filtrates by ether extraction and resolved by thin-layer chromatography. Toxin formation in quiescent cultures was enhanced by malonate but was not markedly affected by ethyl malonate, shikimate, and acetate or by isocitrate or oxaloacetate added in the presence of malonate. Citrate, cis-aconitate, -ketoglutarate, succinate, fumarate, and malonate when present in the medium alone or in conjunction with malonate caused a 15 to 50% reduction in rubratoxin formation. Acetyl-CoA (10-⁵ M/flask) caused an 80% increase in toxin yield. Rubratoxin formation in shake cultures was not affected by succinate and malonate. All other combinations of intermediates and malonate caused a 10 to 50% reduction in toxin formation. At 10^–3 M, citrate enhanced rubratoxin B formation and stimulated rubratoxin A production by as much as 100%. Above 10^–3 M, citrate inhibited toxin production. Incorporation of [2-¹⁴C]acetate into rubratoxin was enhanced by malonate, fumarate, and malonate. A combination of pyruvate and malonate produced a 40% increase in [2-¹⁴C]acetate incorporation into rubratoxin. The highest reduction of labeled acetate incorporation (36%) was caused by succinate or -ketoglutarate combined with malonate.     

Effect of substrates on the malonate inhibitory pattern and brefeldin a formation inCurvularia lunata

The extracellular level of brefeldin A fluctuates with the length of malonate inhibition. Following treatment with malonate, myeelial multiplication as opposed to brefeldin A formation, was preferentially increased in the maleate, fumarate, succinate, citrate, methyl palmitate and glucose replacement cultures. Competitive maleate-malonate, fumarate — malonate, succinate — malonate and citrate-mal-onate-inhibited replacement cultures gave significantly higher mycelial and brefeldin A yields than the sole malonate-inhibited replacement cultures.     

Histochemical modification of the active site of succinate dehydrogenase with N-acetylimidazole

The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.     

Biosynthesis of malonate in roots of soybean seedlings

Many plants accumulate malonate, but it was shown earlier that malonate does not accumulate as a deadend product of metabolism in soybean (Glycine max v. Hodgson tissues. The metabolism of malonate in the soybean plant at the whole tissue and enzymic level was followed, and the pathway of malonate biosynthesis in young soybean root tissue was shown to be via acetyl-coenzyme A carboxylase.     

Enzymic and genetic basis for bacterial growth on malonate

Various bacteria are able to grow aerobically or anaerobically on malonate as sole source of carbon and energy. Independent of the mechanism for energy conservation, the decarboxylation of malonate is the key reaction in the decomposition of this compound. To achieve malonate decarboxylation under physiological conditions, the substrate must be converted into an activated (thioester) derivative. We report here on the malonate decarboxylases of Malonomonas rubra and Klebsiella pneumoniae. These enzymes perform an interesting substrate activation mechanism by generating a malonyl thioester with the enzyme. Formation of the malonyl-S-enzyme involves an 'activation module' that comprises the acetylation of a specific thiol group of an acyl carrier protein (ACP) and the transfer of the ACP moiety to malonate, yielding malonyl-S-ACP and acetate. The malonyl-S-ACP is subsequently decarboxylated with regeneration of the acetyl-ACP. The malonate activation mechanism is related to the activation of citrate by citrate lyase. The relationship extends to the identical 2'-(5'-phosphoribosyl)-3'-dephospho-CoA thiol cofactor that is bound covalently to the corresponding ACP subunit. In Klebsiella pneumoniae, malonate is decarboxylated by a water-soluble enzyme complex. In the anaerobic bacterium Malonomonas rubra, malonate decarboxylation is catalysed by a set of water-soluble as well as membrane-bound enzymes that function together in converting the free energy of the decarboxylation reaction into delta muNa+. Therefore, this malonate decarboxylase includes a biotin carrier protein that accepts the CO2 moiety from malonyl-S-ACP and delivers it to a membrane-bound decarboxylase acting as a Na+ pump. Genes encoding the individual protein components that perform the decarboxylation of malonate in K. pneumoniae or M. rubra have been identified within the mdc and mad gene clusters respectively. The function of most of the derived proteins could be envisaged from sequence similarities with proteins of known functions. The genetic evidence firmly supports the idea that malonate decarboxylation is carried out by the two different decarboxylases, as deduced from the biochemical studies of the enzymes.     

Organic Acid contents of soybean: age and source of nitrogen

The organic acid content of soybean (Glycine max v. Hodgson) root, stem, and leaf tissue was followed for 33 days after germination. Malonate was the predominant acid in leaf and root tissue, whereas fumarate was predominant in the stem. The malonate concentrations of the stem and root showed similar variations with time, but the leaf response was quite different. In nodules from 33-day-old plants, malonate was the predominant acid. Malonate levels in root and nodule tissue of 33-day-old plants were depressed in response to the addition of either nitrate or ammonia. Nodule tissue had a higher malonate concentration on nitrate nitrogen than it did on ammonium nitrogen, whereas root tissue had the higher malonate concentration on ammonium nitrogen. Analysis of organic acid concentrations of roots as a function of age and distance from the root tip in young soybean seedlings revealed a zone consistently high in malate. The malonate level in the entire root rose dramatically in tissue of age 96 to 120 hours.     

Striatal Damage and Oxidative Stress Induced by the Mitochondrial Toxin Malonate Are Reduced in Clorgyline-Treated Rats and MAO-A Deficient Mice

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.     

Symbiotic effects of deltamatB Rhizobium leguminosarum bv. trifolii mutant on clovers

The role of malonate in symbiotic nitrogen metabolism has long been controversial, although it is known to occur in legume roots, especially in the nodules. Here we report that malonate metabolism plays a key role in the differentiation of bacteroids Rhizobium leguminosarum bv. trifolii in clover nodules. An operon, mat, that consists of three consecutive genes (matABC) has been discovered. Mat encodes enzymes that catalyze the uptake and conversion of malonate to acetyl-CoA through malonyl-CoA. A mutant bacteria, which replaced matB that encodes malonyl-CoA synthetase with a kanamycin resistant gene, was generated and infected with white clover. Clover growth was considerably reduced, even though nodules were formed. However, the nodules were filled with vacuoles, but not with bacteroids. This indicates that malonate metabolism is an important requirement for the formation of mature nodules that are filled with bacteroids.     

Low virulence of a morphological Candida albicans mutant

The DNA fragment encoding malonate decarboxylase, involved in malonate assimilation, was cloned from Pseudomonas putida. The 11-kb DNA fragment contained nine open reading frames, which were designated mdcABCDEGHLM in the given order. N-terminal protein sequencing established that the mdcA, mdcC, mdcD, mdcE and mdcH genes encoded subunits alpha, delta, beta, gamma and epsilon of the malonate decarboxylase, respectively. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmid harboring the entire gene cluster or the mdc genes lacking the mdcL and mdcM genes. The mdcL and mdcM genes encode membrane proteins and disruption of the genes of P. putida by the insertion of a kanamycin resistance cassette reduced the malonate uptake activity of the organism. Thus, we conclude that MdcLM is a malonate transporter.     

Effects of malonate administration on renal ammoniagenesis in intact dogs

In the dog kidney in vivo, malonate augmented ammoniagenesis from both amide and nonamide nitrogen sources, similar to previous in vitro investigations using incubating canine renal tubules. This was highly significant in alkalotic dogs, where it was accompanied by decreased renal tissue concentrations of glutamate. Changes in renal ammonia metabolism were less evident in acidotic dogs where a markedly decreased glomerular filtration rate was noted following malonate administration. Under conditions of complete ureteric obstruction which effectively abolished glomerular filtration, malonate significantly augmented ammoniagenesis above baseline in acidotic dogs. These in vivo results with malonate have similarities to those seen in dogs subjected to an acid challenge alone and suggest that the adaptation in renal ammoniagenesis under both circumstances occurs via enhanced deamination of glutamate pools.     

Anaerobic malonate decarboxylation by Citrobacter diversus

Citrobacter diversus ATCC 27156 was able to grow by decarboxylation of malonate to acetate under strictly anaerobic conditions, in the presence of yeast extract. The growth yield, corrected for growth on yeast extract, was 2.03 g cell dry mass per mol malonate. The addition of malonate to ATP-depleted cell suspensions (less than 0.2 nmol ATP/mg cell protein) resulted in a rapid increase in cellular ATP levels to between 4.5 and 6.0 nmol/mg cell protein. Intact cells decarboxylated malonate at rates of up to 1.5 mumol/min.mg protein. Enzyme assays on malonate-grown cells indicated activation of malonate by an ATP-dependent ligase reaction and by CoA transfer from acetyl-CoA, followed by decarboxylation of malonyl-CoA to acetyl-CoA with subsequent recovery of the invested ATP by substrate level phosphorylation through the activity of acetate kinase. Net ATP synthesis is postulated to be mediated by gradient formation coupled to the decarboxylation of malonyl-CoA. The protonophore CCCP and H(+)-ATPase inhibitor DCCD significantly reduced cellular ATP levels, suggesting a role for proton gradients in the energy metabolism of this strain when growing an malonate. Inhibitors of sodium metabolism or ommission of sodium had no effect on ATP levels or malonate decarboxylation.     

Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.     

The origin of free brain malonate

Rat brain contains substantial concentrations of free malonate (192 nmol/g wet weight) but origin and biological importance of the dicarboxylic acid are poorly understood. A dietary source has been excluded. A recently described malonyl-CoA decarboxylase deficiency is associated with malonic aciduria and clinical manifestations, including mental retardation. In an effort to study the metabolic origin of free malonate, several labeled acetyl-CoA precursors were administered by intracerebral injection. [2-¹⁴C]pyruvate or [1,5-¹⁴C]citrate produced radioactive glutamate but failed to label malonate. In contrast, [1-¹⁴C]acetate, [2-¹⁴C]acetate, and [1-¹⁴C]butyrate were converted to labeled glutamateand malonate after the same route of administration. The intracerebral injection of [1-¹⁴C]--alanine as a precursor of malonic semialdehyde and possibly free malonate did not give rise to radioactivity in the dicarboxylate. The labeling pattern of malonic acid is compatible with the reaction sequence: acetyl-CoAmalonyl-CoAmalonate. The final step is thought to occur by transfer of the CoA-group from malonyl-CoA to succinate and/or acetoacetate. Labeling of malonate from acetate is most effective at the age of 7 days when the net concentration of the dicarboxylic acid in rat brain is still very low. At this age, butyrate was a better precursor of malonate than acetate. It is proposed that fatty acid oxidation provides the acetyl-CoA which functions as the precursor of free brain malonate. Compartmentation of malonate biosynthesis is likely because the acetyl-CoA precursors citrate and pyruvate are ineffective.Presented before the 12th Biennial Meeting of the International Society for Neurochemistry, Algarve, Portugal, April 24, 1989.     

Stereochemical course of biotin-independent malonate decarboxylase catalysis.

Malonate decarboxylases, which catalyze the conversion of malonate to acetate, can be classified into biotin-dependent and biotin-independent enzymes. In order to reveal the stereochemical course of the reactions catalyzed by the biotin-independent enzymes from Acinetobacter calcoaceticus and Pseudomonas fluorescens, a chiral substrate, malonate carrying (13)C in one carboxyl group and (3)H at one of the methylene positions, was prepared and used in the reactions catalyzed by these two enzymes. The decarboxylation of (R)-[1-(13)C(1), 2-(3)H]malonate in (2)H(2)O gave a pseudo-racemate of chiral acetate which was converted via acetyl-CoA into malate with malate synthase. From the relative proportions of the isotopomers of malate present, determined by (3)H NMR analysis, it was concluded that in the decarboxylation of malonate by these two biotin-independent enzymes COOH is replaced by H with retention of configuration. The same stereochemical outcome had been previously observed for the reaction catalyzed by the biotin-dependent malonate decarboxylase from Malonomonas rubra (J. Micklefield et al. J. Am. Chem. Soc. 117, 1153-1154, 1995).     

Identification of an Na+-Dependent Malonate Transporter of Malonomonas rubra and Its Dependence on Two Separate Genes

Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na⁺ dependent, and imposition of a ΔpNa⁺ markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into E. coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate⁻ is the transported malonate species. The stimulation of malonate uptake by Na⁺ ions showed Michaelis-Menten kinetics, and a K_m for Na⁺ of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na⁺/Hmalonate⁻ symporter and that it is dependent on two separate genes.     

Malonate as a precursor in the biosynthesis of aflatoxins.

Incorporation of [I-14C]acetate and [2-14C]malonate into aflatoxins by resting mycelia of Aspergillus parasiticus resuspended in different buffers was studied. A decrease in pH from 5-8 to 2-8, as well as addition of EDTA, markedly stimulated the incorporation of malonate but the effect on acetate incorporation was less pronounced. Mycelia took up comparatively more acetate than malonate, but more malonate (4-3%) entering mycelia was incorporated into aflatoxins than was acetate (1-6%). Furthermore, the addition of unlabelled acetate reduced the incorporation of label from [I-14C]acetate by 75% but from [2-14C]malonate by only 25%. These results suggest that malonate is an intermediate in aflatoxin synthesis and that is can be incorporated without prior conversion to acetate.