Four-carbon Dicarboxylic Acid Research Articles

Pioneering research on C<sub>4</sub> leaf anatomical, physiological, and agronomic characteristics of tropical monocot and dicot plant species: Implications for crop water relations and productivity in comparison to C<sub>3</sub> cropping systems

The review is done to summarise the history of the discoveries of the many anatomical, agronomical, and physiological aspects of C4 photosynthesis (where the first chemical products of CO2 fixation in illuminated leaves are four-carbon dicarboxylic acids) and to document correctly the scientists at the University of Arizona and the University of California, Davis, who made these early discoveries. The findings were milestones in plant science that occurred shortly after the biochemical pathway of C3 photosynthesis in green algae (where the first chemical product is a three-carbon compound) was elucidated at the University of California, Berkeley, and earned a Nobel Prize in chemistry. These remarkable achievements were the result of ground-breaking pioneering research efforts carried out by many agronomists, plant physiologists and biochemists in several laboratories, particularly in the USA. Numerous reviews and books written in the past four decades on the history of C4 photosynthesis have focused on the biochemical aspects and give an unbalanced history of the multidisciplinary/multinstitutional nature of the achievements made by agronomists, who published much of their work in Crop Science. Most notable among the characteristics of the C4 species that differentiated them from the C3 ones are: (I) high optimum temperature and high irradiance saturation for maximum leaf photosynthetic rates; (II) apparent lack of CO2 release in a rapid stream of CO2-free air in illuminated leaves in varying temperatures and high irradiances; (III) a very low CO2 compensation point; (IV) lower mesophyll resistances to CO2 diffusion coupled with higher stomatal resistances, and, hence, higher instantaneous leaf water use efficiency; (V) the existence of the so-called “Kranz leaf anatomy” and the higher internal exposed mesophyll surface area per cell volume; and (VI) the ability to recycle respiratory CO2 by illuminated leaves.

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05 wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.

Metabolic flux analysis of a glycerol-overproducingSaccharomyces cerevisiaestrain based on GC-MS, LC-MS and NMR-derived13C-labelling data

This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.

Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.

Fumarate, Maleate, and Succinate Adsorption on Hydrous δ-Al2O3. 2. Electrophoresis Observations and Ionic-Strength Effects on Adsorption

Although the four-carbon dicarboxylic acids (fumarate, maleate, and succinate) can be adsorbed specifically on hydrous δ-Al2O3, the isoelectric point (IEP) was not observed to shift in the three adsorption systems studied due to insignificant adsorption near the IEP and low intrinsic binding affinity of these acids for the oxide surface. Evidence of low intrinsic affinity arose from the strong ionic-strength dependence of the adsorption behavior for each of the three FCDAs. The ionic-strength effects on the adsorption behavior found for maleate were close to or slightly stronger than those of succinate, while fumarate exhibited the strongest ionic-strength dependence among the three FCDAs. These results were well related to their respective adsorption maxima (under-saturation) investigated in part 1. Therefore, it is suggested that, under the same experimental conditions, the adsorption of a particular anion will be attributed to specific adsorption if the ionic-strength effects of this anion are weaker than any of the three FCDAs studied. In addition, the lack of a shift in the isoelectric point cannot be used for judging whether the adsorption is nonspecific.

Fumarate, Maleate, and Succinate Adsorption on Hydrous δ-Al2O3. 1. Comparison of the Adsorption Maxima and Their Significance

Adsorption behaviors among fumarate, maleate, and succinate were compared using the same adsorbent δ-Al2O3 and initial adsorbate concentrations. The adsorption maxima were found all near the midpoint of their respective two pK values indicating that the adsorption of HX- was more favorable than adsorption of H2X and X2- and the adsorption densities were significantly influenced by the electrostatic microenvironment. The adsorption maxima of maleate were about 10% higher than those of fumarate but close to or slightly less than those of succinate before saturation was approached. However, when the adsorbent surface became crowded, the saturated adsorption maxima of maleate and succinate were ca. 1/3 and 1/2, respectively, higher than that of fumarate, indicating that the trans-form isomer occupied more binding space than the cis-form and the single bond anion (succinate) occupied the least binding space among the three four-carbon dicarboxylic acids due to its molecular flexibility.

Purification process for succinic acid produced by fermentation

Succinic acid is a versatile four-carbon dicarboxylic acid. It can be used commerically as an intermediate chemical for the manufacture of 1,4-butanediol, maleic anhydride, and many other chemicals. Succinic acid can be produced by the fermentation of carbohydrates. A complete process for the production and purification of succinic acid from carbohydrates has been developed. The process includes fermentation, desalting electrodialysis, water-splitting electrodialysis, and crystallization to produce a pure crystalline succinic acid. This article will present experimental work performed in the development of this process.

Inhibitory Potential of Four-Carbon Dicarboxylic Acids on Clostridium botulinum Spores in an Uncured Turkey Product

Organic acids offer promising options for the food industry in its attempt to ensure product safety and to meet consumer demand for minimally processed foods. In this study, four-carbon dicarboxylic acids were individually screened for their inhibitory potential against proteolytic Clostridium botulinum spores. Ground turkey breast meat was formulated with 1.4% sodium chloride (NaCl), 0.3% sodium pyrophosphate, 2% organic acid, 8% water and 500 spores/g of a six-strain mixture of proteolytic C. botulinum. Samples were adjusted to pH 6. Ten g of product in vacuum packages were heated in 75°C water for 20 min, cooled and incubated for 0 to 25 days at 28°C. Botulinal neurotoxin was detected at two days in control samples (0% acid) and at five days in 2% malic acid (0.13 M), aspartic (0.13 M), tartaric (0.12 M), succinic (0.15 M), fumaric (0.15 M) samples. Toxin was undetected at 25 days in samples treated with maleic acid (0.15 M). Maleic acid reduced total aerobic bacteria and lactic acid organisms in temperature-abused product, compared to controls. Further systematic investigation of these and related compounds with prior approval for food-use may demonstrate previously unrecognized antibacterial potential.

Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus

The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO(3)). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.

Degradation of malathion by salt-marsh microorganisms

Numerous bacteria from a salt-marsh environment are capable of degrading malathion, an organophosphate insecticide, when supplied with additional nutrients as energy and carbon sources. Seven isolates exhibited ability (48 to 90%) to degrade malathion as a sole carbon source. Gas and thin-layer chromatography and infrared spectroscopy confirmed malathion to be degraded via malathion-monocarboxylic acid to the dicarboxylic acid and then to various phosphothionates. These techniques also identified desmethyl-malathion, phosphorthionates, and four-carbon dicarboxylic acids as degradation products formed as a result of phosphatase activity.

Evidence from 13C NMR for protonation of carbamyl-P and N-(phosphonacetyl)-L-aspartate in the active site of aspartate transcarbamylase.

Nuclear magnetic resonance has been used to study the binding of [13C]carbamyl-P (90% enriched) to the catalytic subunit of Escherichia coli aspartate transcarbamylase. Upon forming a binary complex, there is a small change in the chemical shift of the carbonyl carbon resonance, 2 Hz upfield at pH 7.0, indicating that the environments of the carbonyl group in the active site and in water are similar. When succinate, an analog of L-aspartate, is added to form a ternary complex, there is a large downfield change in the chemical shift for carbamyl-P, consistent with interaction between the carbonyl group and a proton donor of the enzyme. The change might also be caused by a ring current froma nearby aromatic amino acid residue. From the pH dependence of this downfield change and from the effects of L-aspartate analogs other than succinate, the form of the enzyme involved is proposed to be an isomerized ternary complex, previously observed in temperature jump and proton NMR studies. The downfield change to chemical shift for carbamyl-P bound to the isomerized complex is 17.7 +/- 1.0 Hz. Using this value, the relative ability of other four-carbon dicarboxylic acids to form isomerized ternary complexes with the enzyme and carbamyl-P has been evaluated quantitatively. The 13C peak for the transition state analog N-(phosphonacetyl)-L-aspartate (PALA), 90% enriched specifically at the amide carbonyl group, is shifted 20 Hz downfield of the peak for free PALA upon binding to the catalytic subunit at pH 7.0. In contrast, the peak for [1-13C] phosphonaceatmide shifts upfield by about 6 Hz upon binding. Since PALA induces isomerization of the enzyme and phosphonacetamide does not, these data provide further evidence consistent with protonation of the carbonyl group only upon isomerization. The degrees of protonation is strong acids of the carbonyl groups of PALA, phosphonacetamide and urethan (a model for the labile carbamyl-P) have been determined, as have the chemical shifts for these compounds upon full protonation. From these data it is calculated that the amide carbonyl groups of carbamyl-P and PALA might be protonated to a maximum of about 20% in the isomerized complexes at pH 7.0. The change in conformation of the enzyme-carbamyl-P complex upon binding L-aspartate, previously proposed to aid catalysis by compressing the two substrates together in the active site, may be accompanied by polarization of the C=O bond, making this ordinarily unreactive group a much better electrophile. A keto analog of PALA, 4,5-dicarboxy-2-ketopentyl phosphonate, also binds tightly to the catalytic subunit and induces a very similar conformational change, whereas an alcohol analog, 4,5-dicarboxy-2-hydroxypentyl phosphonate, does not bind tightly, indicating the critical importance of an unhindered carbonyl group with trigonal geometry.

Watering converts a CAM plant to daytime CO2 uptake

THREE different photosynthetic options have been identified in plants1,2: (1) most plants have the reductive pentose phosphate or C3 pathway, where CO2 is incorporated into ribulose-1,5-diphosphate (RuDP) to yield two molecules of 3-phosphoglyceric acid, a three-carbon compound; (2) the C4 mode, where the first photosynthetic products are four-carbon dicarboxylic acids like oxaloacetate and malate formed following CO2 incorporation into phosphoenolpyruvate (PEP); and (3) crassulacean acid metabolism (CAM), found in many succulent plants growing in arid regions. In the last, stomatal opening and net CO2 uptake occur at night, CO2 being incorporated by way of PEP carboxylase into organic acids. The tissue acidity decreases as the organic acids are decarboxylated during the day, when the internally released CO2 is prevented from leaving by the closed stomata. The water vapour concentration difference between the tissue and ambient air is less at night, and thus the night-time stomatal opening of CAM plants leads to overall water conservation. For example, the water lost per CO2 fixed averages about sixfold higher for C4 plants and tenfold higher for C3 ones than for CAM plants in natural conditions2. The net daily CO2 uptake by CAM plants is less than for C3 or C4 plants, so CAM plants tend to be relatively slow growing.

Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (p K a 's of 6.9 and 8.6). The pH dependence of the p K i for succinate, a product of isocitrate cleavage, implied that a dissociable group (p K a of 6.0) on the enzyme functions in binding succinate. The p K i 's for maleate and itaconate (succinate analogs) were similarly pH dependent. The p K i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the p K i 's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the p K m for isocitrate and the p K i for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (p K a of 5.8). The pH dependence of the p K i for homoisocitrate was similar. In addition the K i for succinate and K m for isocitrate were dependent upon Mg 2+ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (p K a of 5.8) and one on phosphoenolpyruvate (p K a of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate.

Escherichia coli phosphoenolpyruvate carboxylase: Effect of allosteric inhibitors on the kinetic parameters and sedimentation behavior

Various metabolic intermediates and chemically related compounds were studied to determine their effects on the catalytic activity and sedimentation behavior in sucrose gradients of phosphoenolpyruvate carboxylase of Escherichia coli. Two types of enzyme-inhibitor interactions were found. In the first type of inhibition, the inhibitor was functionally competitive with acetyl coenzyme A and had the ability to stabilize the tetrameric form of the enzyme, as evidenced by its effect on the apparent sedimentation rate of the enzyme in sucrose gradients. These compounds were approximately equivalent in length to four-carbon dicarboxylic acids and had to be ionized with negative charges at both ends. In addition, the strong inhibitors (aspartate, malate, fumarate, cysteine, and tartrate) have an electron-attracting group as an L-α substituent. Fumarate was also a strong inhibitor, and it too contained an electron-attracting group in the form of an olefinic bond. The second type of inhibition was exhibited uniquely by maleate. Maleate inhibited the enzyme strongly, but it was not competitive with acetyl coenzyme A nor could it stabilize the tetrameric form of the enzyme at concentrations that were highly inhibitory. The pattern of inhibition by maleate could be converted to that seen with the other inhibitors in the presence of aspartate. This indicates that each class of inhibitor is competitive with the other and that all allosteric inhibitors may share the same binding site.

Use of 13C in biosynthetic studies. Incorporation of isotopically labeled acetate and aspartate into fusaric acid.

Acetate-1-13C, acetate-2-13C, and aspartate-4-13C were used as substrates to elucidate the biosynthetic pathway for fusaric acid, the phytotoxic metabolite of Fusarium oxysporum Schlecht. It was established that C-2, C-3, C-4, and C-7 were derived from acetate via aspartate or a related four-carbon dicarboxylic acid, whereas C-5, C-6, C-8, C-9, C-10, and C-11 were derived more directly from acetate. The 13C-labeling patterns were determined by measuring the relative intensities of the 13C—H satellites by proton magnetic resonance spectroscopy.Further information was derived from experiments with D- and L-isomers of aspartate-1-14C and L-aspartate-1-14C:15N. Radioactivity from both enantiomers of aspartate-1-14C was incorporated into fusaric acid, but more efficiently from the L-isomer. Furthermore, 15N was incorporated more efficiently into fusaric acid and the aspartic acid of mycelial protein than was 14C. These results suggest that aspartic acid is metabolized to fusaric acid via oxalacetate, and that L-aspartate serves as a donor of nitrogen, in an amino-transferase reaction, to a separate oxalacetate pool of primarily endogenous origin.

Inactivation of citrate lyase by oxaloacetate and its structural analogues.

Abstract 1. 1. The inactivation of citrate lyase (citrate-oxaloacetate lyase, EC 4.1.3.6) by oxaloacetate has been investigated. 2. 2. Studies of the pH profiles of inactivation at pH 7–9 with varying total oxaloacetate and magnesium concentrations show that the magnesium complexes of enolic oxaloacetate are involved. 3. 3. The inhibitory effects of the following structural analogues of the keto and enol forms of oxaloacetate were examined: l -malate, α,α-dimethyloxaloacetate, tartronate, α,α-difluorooxaloacetate, pyruvate, ketomalonate, and isomalate. These results, and the effect of other divalent metal cations, indicate that the site of the inactivation is identical with the active site for citrate cleavage. 4. 4. The inactivation is irreversible and is time and concentration dependent. Free oxaloacetate does not inactivate the enzyme. 5. 5. The inactivation phenomenon has high structural specificity, requiring a straight-chain, four-carbon dicarboxylic acid with an ionisable α-hydroxy group, and the presence of a divalent metal cation.

Niacin Metabolism in Mycobacteria1,2

Human tubercle bacilli, as compared with other mycobacteria, produce a large amount of in the culture medium (1). The niacin (2), which distinguishes human tubercle bacilli biochemically from other mycobacteria, is based on this difference in production. Albertson and Moat (3) reported that niacin-14C was formed from aspartic acid-14C and suggested that was formed by the condensation of a four-carbon dicarboxylic acid with a three-carbon compound. Konno and co-workers (4, 5), using cellfree extracts of mycobacteria, reported that quinolinic acid was a precursor of ribonucleotide (deamido-NMN). The enzyme activity that converts quinolinic acid to deamidoNMN was found to parallel production. Nakamura, Nishizuka, and Hayaishi (6), using a purified beef liver extract system, demonstrated the regulation of nicotinamide adenine dinucleotide (NAD) biosynthesis by adenosine triphosphate (ATP). In the absence of ATP, deamido-NMN is converted to ribonucleoside and eventually to niacin. However, when a sufficient amount of ATP is available, and ribonucleoside are utilized for the synthesis of deamido-NMN, and no accumulates. The production of excess by human strains of mycobacteria suggested that a similar mechanism might be operative, and the studies reported here were designed to test this hypothesis.

THE ORIGIN OF CARBON ATOMS 2, 3, AND 7 OF RICININE

The isolation of carbon atoms 2 and 3 of the α-pyridone nucleus of ricinine is described. Evidence is provided to support the suggestion that succinic acid or a closely related four-carbon dicarboxylic acid provides carbon atoms 2, 3, and 7 in the biosynthesis of the ricinine molecule.