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PubMed1999

Effect of the addition of fumarate on methane production by ruminal microorganisms in vitro.

The effect of fumarate used as a feed additive on the reduction of methanogenesis in the rumen was evaluated by in vitro experiments. The addition of fumarate to the culture of mixed ruminal microorganisms that were fermenting hay powder and concentrate reduced methane production. Most fumarate was metabolized to propionate, and a slight increase was noted in other volatile fatty acids. Fumarate was utilized by mixed bacteria but not by mixed protozoa. Fibrobacter succinogenes, Selenomonas ruminantium ssp. ruminantium, Selenomonas ruminantium ssp. lactilytica, Veillonella parvula, and Wollinella succinogenes oxidized H2 by using fumarate as a final electron acceptor, suggesting that these bacteria compete with methanogens for H2, which is the main substrate for methanogenesis in the rumen. However, the affinity of these bacteria to H2 was lower than their affinity to methanogens. These fumarate-utilizing bacteria metabolized malate to products that were similar to those from fumarate, suggesting the possession of fumarate dehydratase. Fibrobacter succinogenes, V. parvula, and W. succinogenes utilized formate, another substrate for methanogenesis, as an electron donor for fumarate reduction. The affinity of these bacteria to formate was higher than the affinity methanogenes have for formate. When methanogens were cocultured with an equal cellular amount of each of the fumarate-utilizing bacteria, methane production was markedly decreased, not only from formate, but also from H2. These results suggest that the addition of fumarate to ruminant feed reduces methanogenesis and enhances propionate production in the rumen.

Adenosine Triphosphate↗

PubMed1999

Influence of sodium fumarate addition on rumen fermentation in vitro.

The influence of sodium fumarate on rumen fermentation was investigated in vitro using batch and semi-continuous cultures of mixed rumen micro-organisms taken from three sheep receiving a basal diet of hay, barley, molasses, fish meal and a mineral-vitamin supplement (500, 299.5, 100, 91 and 9.5 g/kg DM respectively). Batch cultures consisted of 10 ml strained rumen fluid in 40 ml anaerobic buffer containing 200 mg of the same feed given to the sheep. Sodium fumarate was added to achieve a final concentration of 0, 5 or 10 mmol/l, as a result of the addition of 0, 250 or 500 mumol, equivalent to 0, 200 and 400 g/kg feed. CH4 production at 24 h (360 mumol in the control cultures) fell (P < 0.05) by 18 and 22 mumol respectively (SED 7.5). Total gas production was increased by the addition of fumarate without significant accumulation of H2. Substantial increases in acetate production (92 and 194 mumol; SED 26.7, P < 0.01) were accompanied by increases in propionate formation (212 and 396 mumol; SED 13.0, P < 0.001). Longer-term effects of fumarate supplementation on ruminal fermentation and CH4 production were investigated using the rumen simulation technique (Rusitec). Eight vessels were given 20 g basal diet/d, and half of them received a supplement of fumarate (disodium salt) over a period of 19 d. The response to the daily addition of 6.25 mmol sodium fumarate was a decrease in CH4 production of 1.2 mmol (SED 0.39, P < 0.05), equivalent to the consumption of 4.8 mmol H2, and an increase in propionate production of 4.9 mmol (from 10.4 to 15.3 (SED 1.05) mmol/d, P < 0.01). The inhibition of CH4 production did not decline during the period of time that fumarate was added to the vessels. Thus, the decrease in CH4 corresponded well to the fraction of the fumarate that was converted to propionate. Fumarate had no significant (P > 0.05) effect on total bacterial numbers or on the number of methanogenic archaea, but numbers of cellulolytic bacteria were increased (8.8 v. 23.9 (SED 2.49) x 10(5) per ml, P < 0.01). Fumarate also increased DM digestibility of the basal diet after 48 h incubation (0.476 v. 0.508 (SED 0.0123), P < 0.05). Thus, it was concluded that sodium fumarate may be a useful dietary additive for ruminants, because it diverts some H2 from CH4 production and because it is able to stimulate proliferation of cellulolytic bacteria and digestion of fibre.

Acetates↗

PubMed1993

Sensory rhodopsin-controlled release of the switch factor fumarate in Halobacterium salinarium.

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.

Cell Membrane↗

PubMed2003

Identification of the gene encoding the sole physiological fumarate reductase in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a Gram-negative, nonfermentative rod with a complex electron transport system which facilitates its ability to use a variety of terminal electron acceptors, including fumarate, for anaerobic respiration. CMTn-3, a mutant isolated by transposon (TnphoA) mutagenesis, can no longer use fumarate as an electron acceptor; it lacks fumarate reductase activity as well as a 65-kDa soluble tetraheme flavocytochrome c. The sequence of the TnphoA-flanking genomic DNA of CMTn-3 did not align to those for fumarate reductase or related electron transport genes from other bacteria. Sequence analysis of the MR-1 genomic database demonstrated that an open reading frame encoding a 65-kDa tetraheme cytochrome c with sequence similarity to the fumarate reductase from S. frigidimarina NCIMB400 was found 8 kb away from the TnphoA-flanking genomic DNA of CMTn-3. PCR analysis demonstrated that a large deletion (>or=9.2 kb and <or=11 kb) of genomic DNA occurred in CMTn-3 as a result of TnphoA insertion. This deletion included at least half of the fumarate reductase gene as well as approximately 8 kb of upstream DNA. Complementation of CMTn-3 with the fumarate reductase gene plus 0.5-kb of upstream DNA restored growth on fumarate. These studies explicitly define the sole physiological fumarate reductase gene from the several possibilities suggested by the genomic sequence of MR-1. Surprisingly, the fumarate reductase gene plus 0.77-kb upstream DNA from S. frigidimarina NCIMB400 did not complement CMTn-3.

DNA Transposable Elements↗

PubMed1998

Characterization of fumarate transport in Helicobacter pylori.

The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. The transport of fumarate at micromolar concentrations was saturable with a KM of 220 +/- 21 micron and Vmax of 54 +/- 2 nmole/min/mg protein at 20 degrees C, depended on temperature between 4 and 40 degrees C, and was susceptible to inhibitors, suggesting the presence of one or more fumarate carriers. The release of fumarate from cells was also saturable with a KM of 464 +/- 71 micron and Vmax of 22 +/- 2 nmol/min/mg protein at 20 degrees C. The rates of fumarate influx at millomolar concentrations increased linearly with permeant concentration, and depended on the age of the cells. The transport system was specific for dicarboxylic acids suggesting that fumarate is taken up via dicarboxylate transporters. Succinate and fumarate appeared to form an antiport system. The properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential inhibitors. The results provided evidence that influx and efflux of fumarate at low concentrations from H. pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion. The anaerobic C4-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate transporter.

Amino Acids↗

PubMed2002

Plausible molecular mechanism for activation by fumarate and electron transfer of the dopamine beta-mono-oxygenase reaction.

A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine beta-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans -glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1-5 mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine-fumarate conjugate, N -(2-phenylethyl)fumaramide (PEA-Fum), is an approximately 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency approximately 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine alpha-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine beta-mono-oxygenase.

Amides↗

PubMed2003

Influence of different concentrations of disodium fumarate on methane production and fermentation of concentrate feeds by rumen micro-organisms in vitro.

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of disodium fumarate on the fermentation of five concentrate feeds (maize, barley, wheat, sorghum and cassava meal). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mM-fumarate. In 17 h incubations, the final pH and total volatile fatty acid production increased (P<0.001) linearly for all substrates as fumarate concentration increased from 0 to 10 mm. Propionate and acetate production increased (P<0.05), while the value of the acetate:propionate ratio decreased (P<0.05) linearly with increasing doses of fumarate. In contrast, l-lactate and NH3-N concentrations in the cultures were not affected (P>0.05) by the addition of fumarate. For all substrates, fumarate treatment decreased (P<0.05) CH4 production, the mean values of the decrease being 2.3, 3.8 and 4.8 % for concentrations of 4, 7 and 10 mM-fumarate respectively. Addition of fumarate did not affect (P>0.05) the total gas production. If the results of the present experiment are confirmed in vivo, fumarate could be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH, acetate and propionate production and it decreased CH4 production.

Acetates↗

PubMed1998

Regulation of switching frequency and bias of the bacterial flagellar motor by CheY and fumarate.

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of Switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counter-clockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.

Arabinose↗

PubMed1996

L-aspartate oxidase from Escherichia coli. II. Interaction with C4 dicarboxylic acids and identification of a novel L-aspartate: fumarate oxidoreductase activity.

L-Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced L-aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. L-Aspartate oxidase binds succinate and fumarate with Kd values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of L-aspartate oxidase is reported using benzylviologen or L-aspartate as reductants and fumarate as oxidant. Steady-state kinetics for the oxidase and the fumarate reductase activity of L-aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and L-aspartate as electron donor. Finally, succinate was identified as the product of the L-aspartate:fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for L-aspartate oxidase.

Amino Acid Oxidoreductases↗

PubMed1993

Growth of syntrophic propionate-oxidizing bacteria with fumarate in the absence of methanogenic bacteria.

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO(2). Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii, such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.

Journal Article↗

PubMed2005

Effects of disodium fumarate on in vitro rumen microbial growth, methane production and fermentation of diets differing in their forage:concentrate ratio.

The effects of disodium fumarate on microbial growth, CH4 production and fermentation of three diets differing in their forage content (800, 500 and 200 g/kg DM) by rumen micro-organisms in vitro were studied using batch cultures. Rumen contents were collected from four Merino sheep. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4 and 8 mm-fumarate, and (15)N was used as a microbial marker. Gas production was measured at regular intervals from 0 to 120 h of incubation. Fumarate did not affect (P>0.05) any of the measured gas production parameters. In 17 h incubations, the final pH and the production of acetate and propionate were increased linearly (P<0.001) by the addition of fumarate. Fumarate tended to increase (P=0.076) the organic matter disappearance of the diets and to decrease (P=0.079) the amount of NH3-N in the cultures. Adding fumarate to batch cultures tended (P=0.099) to decrease CH4 production, the mean values of the decrease being 5.4 %, 2.9 % and 3.8 % for the high-, medium- and low-forage diet, respectively. Fumarate tended to increase (P=0.082) rumen microbial growth for the high-forage diet, but no differences (P>0.05) were observed for the other two diets. These results indicate that the effects of fumarate on rumen fermentation depend on the nature of the incubated substrate, the high-forage diet showing the greatest response.

Acetates↗

PubMed1999

Effect of fumaric acid and dietary buffering capacity on ileal and fecal amino acid digestibilities in early-weaned pigs.

Experiments were conducted to determine the effect of fumaric acid supplementation and buffering capacity of the diet on ileal and fecal digestibilities of CP, GE, and amino acids in early-weaned pigs. In two experiments, 12 pigs each were weaned at 14 d of age and fitted with a simple T-cannula at the distal ileum between 15 and 17 d of age. In both experiments, the pigs were fed four diets, based on wheat and soybean meal without (control) or with 1, 2, or 3% fumaric acid according to a balanced two-period change-over design. In Exp. 2, the buffering capacity of the diets was increased by supplementation of 3% sodium bicarbonate. The pigs were fed three times daily: equal amounts at 8-h intervals. The diets were supplied at a rate of 5% (wt/wt) of body weight. The inclusion of fumaric acid to the diet with a low buffering capacity increased (P < .05) the ileal digestibilities of CP, GE, and the majority of amino acids. The highest responses were found at an inclusion level of 2% fumaric acid. The improvements in apparent ileal amino acid digestibilities ranged from 4.9 to 12.8 percentage units. Supplementation of fumaric acid to a diet with a high buffering capacity led only to numerical increases in ileal digestibilities of CP, GE, and amino acids. In both studies, fumaric acid supplementation had no effect (P > .05) on the fecal digestibilities of CP, GE, and amino acids, except histidine. A high buffering capacity of the diet decreased the ileal amino acid digestibilities by 1 to 10 percentage units compared with diets with the low buffering capacities. In both experiments, ileal and fecal digestibilities were higher (P < .05) in Period 2 (on d 24 after weaning) than in Period 1 (on d 11 after weaning). A positive correlation (P < .05) between ileal digestibilities and fumaric acid supplementation to the diets with the low buffering capacities was observed in Period 1 (on d 11 after weaning), but not in Period 2 (on d 24 after weaning). No relationship (P > .05) between ileal digestibilities and fumaric acid supplementation to the diets with a high buffering capacity could be established. In conclusion, supplementation of fumaric acid to starter diets during the first 3 to 4 wk after weaning increases the ileal digestibilities of GE, CP, and amino acids.

Amino Acids↗

PubMed1990

Induction of glutathione transferases and NAD(P)H:quinone reductase by fumaric acid derivatives in rodent cells and tissues.

Dimethyl fumarate and dimethyl maleate are potent inducers of cytosolic NAD(P)H:(quinone acceptor) oxidoreductase (here designated quinone reductase) activity in Hepa 1c1c7 murine hepatoma cells in culture, whereas fumaric and maleic acids are much less potent, in agreement with the much greater reactivity of the esters as Michael reaction acceptors (P. Talalay, M. J. De Long, and H. J. Prochaska, Proc. Natl. Acad. Sci. USA, 85:8261-8265, 1988). Dimethyl fumarate also induced quinone reductase in mutants of the Hepa 1c1c7 cell line that were either defective in the Ah receptor or in cytochrome P1-450 activity, thereby establishing that this compound is a monofunctional inducer (H. J. Prochaska and P. Talalay, Cancer Res., 48: 4776-4782, 1988). Addition of dimethyl fumarate to the diet of female CD-1 mice and female Sprague-Dawley rats at 0.2-0.5% concentrations elevated cytosolic glutathione transferases and quinone reductase activities in a variety of organs, whereas much higher concentrations of fumaric acid were only marginally active. The widespread induction of such detoxication enzymes by dimethyl fumarate suggests the potential value of this compound as a protective agent against chemical carcinogenesis and other forms of electrophile toxicity. This proposal is supported by the finding that the concentrations of dimethyl fumarate required to obtain substantial enzyme inductions were well tolerated by rodents. Furthermore, the parent fumaric acid has low chronic toxicity and is a naturally occurring metabolic intermediate that is already in the food chain as an additive, and fumarate salts and esters are used for therapeutic purposes in man.

Animals↗

PubMed2005

Hierarchical control of anaerobic gene expression in Escherichia coli K-12: the nitrate-responsive NarX-NarL regulatory system represses synthesis of the fumarate-responsive DcuS-DcuR regulatory system.

Hierarchical control ensures that facultative bacteria preferentially use the available respiratory electron acceptor with the most positive standard redox potential. Thus, nitrate is used before other electron acceptors such as fumarate for anaerobic respiration. Nitrate regulation is mediated by the NarX-NarL two-component system, which activates the transcription of operons encoding nitrate respiration enzymes and represses the transcription of operons for other anaerobic respiratory enzymes, including enzymes involved in fumarate respiration. These are fumarate reductase (encoded by the frdABCD operon), fumarase B, which generates fumarate from malate, and the DcuB permease for fumarate, malate, and aspartate. The transcription of the corresponding structural genes is activated by the DcuS-DcuR two-component system in response to fumarate or its dicarboxylate precursors. We report results from preliminary transcription microarray experiments that revealed two previously unknown members of the NarL regulon: the aspA gene encoding aspartate-ammonia lyase, which generates fumarate; and the dcuSR operon encoding the dicarboxylate-responsive regulatory system. We measured beta-galactosidase expression from monocopy aspA-lacZ, frdA-lacZ, and dcuS-lacZ operon fusions in response to added nitrate and fumarate and with respect to the dcuR and narL genotypes. Nitrate, acting through the NarX-NarL regulatory system, repressed the transcription of all three operons. Only frdA-lacZ expression, however, was responsive to added fumarate or a dcuR(+) genotype. Phospho-NarL protein protected operator sites in the aspA and dcuS promoter regions from DNase I cleavage in vitro. The overall results are consistent with the hypothesis that nitrate represses frdA operon transcription not only directly, by repressing frdA promoter activity, but also indirectly, by repressing dcuS promoter activity.

Amino Acid Sequence↗

PubMed1964

FUMARATE REDUCTION AND ITS ROLE IN THE DIVERSION OF GLUCOSE FERMENTATION BY STREPTOCOCCUS FAECALIS.

Deibel, R. H. (American Meat Institute Foundation, Chicago, Ill.), and M. J. Kvetkas. Fumarate reduction and its role in the diversion of glucose fermentation by Streptococcus faecalis. J. Bacteriol. 88:858-864. 1964.-Fumarate diverts the normal fermentation of glucose by Streptococcus faecalis FB82, as shown by the production of increased amounts of CO(2), formate, acetate, and acetoin, and decreased formation of lactate and ethanol. Experiments with d-glucose-1-C(14), in which low levels of labeled CO(2) were recovered, indicated that C-1 cleavage of the glucose molecule was not involved. The presence of fumarate afforded consistently larger cell crops in growth studies with glucose and other energy sources. On a molar growth-yield basis, anaerobically grown, glucose-fumarate cultures were equivalent to aerobically grown, glucose cultures. The reduction of fumarate by cell suspensions indicated that glucose, gluconate, and, to a lesser extent, glycerol and mannitol could serve as hydrogen donors. Several common metabolic inhibitors had no effect upon the fumarate reductase system in cell suspensions, although some sensitivity to acidic pH was noted. Significant levels of succinate oxidation activity were not detected. Fumarate reductase activity was demonstrated in all five S. faecalis strains tested. Distribution of this ability in S. faecium strains was variable, ranging from activity comparable with that of S. faecalis to total inactivity. The observations support the conclusion that fumarate functions as an alternate hydrogen acceptor, thus allowing pyruvate to participate in the energy-yielding phosphoroclastic and dismutation pathways.

Acetates↗

PubMed1998

A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens.

During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The DeltafccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor.

Amino Acid Sequence↗

PubMed1992

Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.

The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.

Animals↗

PubMed1996

The specificity of fumarate as a switching factor of the bacterial flagellar motor.

Fumarate restores to flagella of cytoplasm-free, Che Y-containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemotaxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.

Aspartic Acid↗