Malonate metabolism: biochemistry,molecular biology,physiology, and industrial application

Malonate is a three-carbon dicarboxylic acid. It is well known as a competitive inhibitor of succinate dehydrogenase. It occurs naturally in biological systems, such as legumes and developing rat brains, which indicates that it may play an important role in symbiotic nitrogen metabolism and brain development. Recently, enzymes that are related to malonate metabolism were discovered and characterized. The genes that encode the enzymes were isolated, and the regulation of their expression was also studied. The mutant bacteria, in which the malonate-metabolizing gene was deleted, lost its primary function, symbiosis, between Rhizobium leguminosarium bv trifolii and clover. This suggests that malonate metabolism is essential in symbiotic nitrogen metabolism, at least in clover nodules. In addition to these, the genes matB and matC have been successfully used for generation of the industrial strain of Streptomyces for the production of antibiotics.     

Acetylene reduction by effective and ineffective clover and soybean root nodules

Summary Acetylene was reduced to ethylene by effective white clover nodules and by fully and partially effective intact nodules, nodule homogenates, and bacteroids of soybeans. Succinate and several amino acids markedly stimulated the reduction by effective soybean bacteroids, but the stimulation was slight with partially effective bacteroids. Acetylene metabolism by effective soybean bacteroids was also enhanced by excretions of in vitro-grown Rhizobium japonicum, excretions of bacteria derived from effective and ineffective nodules, and the soluble fraction from these nodules. Inhibitors of nitrogen fixation were not found in ineffective nodules. Ineffective soybean and white clover nodules and homogenates or isolated bacteria from ineffective soybean nodules did not reduce acetylene. Additions of succinate, amino acids, the soluble fraction of effective nodules, or excretions of effective bacteroids or of in vitro-grown cells of an effective R. japonicum strain did not promote nitrogen fixation by bacterial cells obtained from ineffective soybean nodules.     

Malonate Catabolism Does Not Drive N2 Fixation in Legume Nodules

Malonyl-coenzyme A (CoA) decarboxylase, malonyl-CoA synthetase, and malonate transporter mutants of Rhizobium leguminosarum bv. viciae and trifolii fixed N₂ at wild-type rates on pea and clover, respectively. Thus, malonate does not drive N₂ fixation in legume nodules.     

The origin of the membrane envelope surrounding the bacteria and bacteroids and the presence of glycogen in clover root nodules

Summary Thin sections of clover nodules were examined by electron microscopy. The emergence of the bacteria from the infection thread was found to occur by a process of phagocytosis. No evidence was found, using glutar-aldehyde-osmium as a fixative, that there is any connection between the membrane envelope and the endoplasmic reticulum.Electron dense granules, previously observed in clover nodule bacteria, were identified as glycogen granules. These glycogen granules accumulated in bacteria and bacteroids in some ineffective nodules.     

Changes in the activity and isozyme patterns of malate dehydrogenase in root nodules of yellow lupine

The malate dehydrogenase present in the cytoplasmic fraction of plant origin and bacteroids from yellow lupine root nodules was investigated. The plant enzyme was 14 times more active in nodules than in roots and it contained 6 molecular forms in nodules compared with 3 forms detected in roots. The highest malate dehydrogenase activity in plant fraction and bacteroids was noted in 50-day old plants. Changes in the isoenzymatic patterns of malate dehydrogenase in plant fraction and bacteroids accompanying ageing of the lupine root nodules were observed. Possible physiological role of malate pathway in metabolism of lupine root nodules is discussed.     

Metabolism of C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules

The metabolism of translocated photosynthate by soybean (Glycine max L. Merr.) nodules was investigated by ¹⁴CO₂-labeling studies and analysis of nodule enzymes. Plants were exposed to ¹⁴CO₂ for 30 minutes, followed by ¹²CO₂ for up to 5 hours. The largest amount of radioactivity in nodules was recovered in neutral sugars at all sampling times. The organic acid fraction of the cytosol was labeled rapidly. Although cyclitols and malonate were found in high concentrations in the nodules, they accumulated less than 10% of the radioactivity in the neutral and acidic fractions, respectively. Phosphate esters were found to contain very low levels of total label, which prohibited analysis of the radioactivity in individual compounds. The whole nodule-labeling patterns suggested the utilization of photosynthate for the generation of organic acids (principally malate) and amino acids (principally glutamate).

The radioactivity in bacteroids as a percentage of total nodule label increased slightly with time, while the percentage in the cytosol fraction declined. The labeling patterns for the cytosol were essentially the same as whole nodule-labeling patterns, and they suggest a degradation of carbohydrates for the production of organic acids and amino acids. When it was found that most of the radioactivity in bacteroids was in sugars, the enzymes of glucose metabolism were surveyed. Bacteroids from nodules formed by Rhizobium japonicum strain 110 or strain 138 lacked activity for phosphofructokinase and NADP-dependent 6-phosphogluconate dehydrogenase, key enzymes of glycolysis and the oxidative pentose-phosphate pathways. Enzymes of the glycolytic and pentose phosphate pathways were found in the cytosol fraction.

In three experiments, bacteroids contained about 10 to 30% of the total radioactivity in nodules 2 to 5 hours after pulse-labeling of plants, and 60 to 65% of the radioactivity in bacteroids was in the neutral sugar fraction at all sampling times. This strongly suggests some absorption and metabolism of sugars by bacteroids in spite of the lack of key enzymes. Bacteroids did possess enzymes for the formation of hexose phosphates from glucose or fructose. Radioactivity in α,α-trehalose in bacteroids increased until, after 5 hours, trehalose was a major labeled compound in bacteroids. Thus, trehalose synthesis may be a major fate of sugars entering bacteroids.

     

Mammalian ACSF3 protein is a malonyl-CoA synthetase that supplies the chain extender units for mitochondrial fatty acid synthesis

The objective of this study was to identify a source of intramitochondrial malonyl-CoA that could be used for de novo fatty acid synthesis in mammalian mitochondria. Because mammalian mitochondria lack an acetyl-CoA carboxylase capable of generating malonyl-CoA inside mitochondria, the possibility that malonate could act as a precursor was investigated. Although malonyl-CoA synthetases have not been identified previously in animals, interrogation of animal protein sequence databases identified candidates that exhibited sequence similarity to known prokaryotic forms. The human candidate protein ACSF3, which has a predicted N-terminal mitochondrial targeting sequence, was cloned, expressed, and characterized as a 65-kDa acyl-CoA synthetase with extremely high specificity for malonate and methylmalonate. An arginine residue implicated in malonate binding by prokaryotic malonyl-CoA synthetases was found to be positionally conserved in animal ACSF3 enzymes and essential for activity. Subcellular fractionation experiments with HEK293T cells confirmed that human ACSF3 is located exclusively in mitochondria, and RNA interference experiments verified that this enzyme is responsible for most, if not all, of the malonyl-CoA synthetase activity in the mitochondria of these cells. In conclusion, unlike fungi, which have an intramitochondrial acetyl-CoA carboxylase, animals require an alternative source of mitochondrial malonyl-CoA; the mitochondrial ACSF3 enzyme is capable of filling this role by utilizing free malonic acid as substrate.     

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.     

A model of nitrogen flow by malonamate in Rhizobium japonicum-soybean symbiosis

Two types of novel malonamidases were found in soybean nodules. One (E1) catalyzes the formation of malonamate from malonate and its hydrolysis to ammonia, whereas the other (E2) acts mainly on the hydrolysis of malonamate. E1 and E2 were found in bacteroids, but only E2 was found in the plant cytosol of the nodule. The substrate requirements of E1 and E2 were highly specific for malonate and malonamate, respectively. From these and other results reported previously, we propose that malonamate plays an important role as a nitrogen carrier in the Rhizobium legume symbiosis.     

Role of malonate in chickpeas

Analysis of the content and distribution of organic acids in chickpea plants (Cicer arietinum L.) showed that malonate was the most abundant acid in roots and nodules, whereas malate was the main acid in leaves and stems. The highest concentration of malonate in roots was in the apices. Malonate metabolism did not appear to be directly related to abiotic stress. We suggest that malonate has a role as a defensive chemical in roots and nodules of chickpeas.     

Malonate, Malonyl-Coenzyme A, and Acetyl-Coenzyme A in Developing Rat Brain

Abstract: Free malonate, malonyl-coenzyme A (malonyl-CoA), and acetyl-CoA were assayed in rat brain at developmental ages from the 20th day of gestation to 60 days of postnatal life. The determination of malonate was based on its conversion to malonyl-CoA and decarboxylation to acetyl-CoA by enzyme extracts from Pseudo-monas fluorescens. The resulting acetyl-CoA reacted with [4-¹⁴C]oxaloacetate to form [5-¹⁴C]citrate, which was isolated by TLC. Malonyl-CoA in perchloric acid extracts from brain was converted to acetyl-CoA by rat liver mitochondrial malonyl-CoA decarboxylase (EC 4.1.1.9). Acetyl-CoA derived from this step was assayed by a modified CoA-cycling procedure. Brain acetyl-CoA was also assayed by CoA cycling. Prenatal brain contained no free malonate but malonyl-CoA was present. The acetyl-CoA level was relatively high just prior to birth and declined slightly with growth. Malonate concentrations after birth rose rapidly to reach 192 nmol/g wet weight at 60 days. Adult levels for malonyl-CoA and acetyl-CoA were 1.83 and 1.90 nmol/g wet weight, respectively. The origin and natural role of free malonate in brain are not known but deacylation of malonyl-CoA by reversal of the malonyl-CoA synthetase reaction is postulated. Rat liver and kidney also contain substantial concentrations of free malonate.     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Effects of Salt Stress on Amino Acid, Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of Alfalfa (Medicago sativa L.)

Ethanol-soluble organic acid, carbohydrate, and amino acid constituents of alfalfa (Medicago sativa) roots and nodules (cytosol and bacteroids) have been identified by gas-liquid chromatography and high performance liquid chromatography. Among organic acids, citrate was the predominant compound in roots and cytosol, with malonate present in the highest concentration in bacteroids. These two organic acids together with malate and succinate accounted for more than 85% of the organic acid pool in nodules and for 97% in roots. The major carbohydrates in roots, nodule cytosol, and bacteroids were (descending order of concentration): sucrose, pinitol, glucose, and ononitol. Maltose and trehalose appeared to be present in very low concentrations. Asparagine, glutamate, alanine, γ-aminobutyrate, and proline were the major amino acids in cytosol and bacteroids. In addition to these solutes, serine and glutamine were well represented in roots. When alfalfa plants were subjected to 0.15 m sodium chloride stress for 2 weeks, total organic acid concentration in nodules and roots were depressed by more than 40%, whereas lactate concentration increased by 11, 27, and 94% in cytosol, roots, and bacteroids, respectively. In bacteroids, lactate became the most abundant organic acid and might contribute partly to the osmotic adjustment. On the other hand, salt stress induced a large increase in the amino acid and carbohydrate pools. Within the amino acids, proline showed the largest increase, 11.3-, 12.8-, and 8.0-fold in roots, cytosol, and bacteroids, respectively. Its accumulation reflected an osmoregulatory mechanism not only in roots but also in nodule tissue. In parallel, asparagine concentration was greatly enhanced; this amide remained the major nitrogen solute and, in bacteroids, played a significant role in osmoregulation. On the contrary, the salt treatment had a very limited effect on the concentration of other amino acids. Among carbohydrates, pinitol concentration was increased significantly, especially in cytosol and bacteroids (5.4- and 3.4-fold, respectively), in which this cyclitol accounted for more than 35% of the total carbohydrate pool; pinitol might contribute to the tolerance to salt stress. However, trehalose concentration remained low in both nodules and roots; its role in osmoregulation appeared unlikely in alfalfa.     

Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules. We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate–glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxin-containing nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed.     

Nitrogen fixation and carbon metabolism in legume nodules

A large amount of energy is utilized by legume nodules for the fixation of nitrogen and assimilation of fixed nitrogen (ammonia) into organic compounds. The source of energy is provided in the form of photosynthates by the host plant. Phosphoenol pyruvate carboxylase (PEPC) enzyme, which is responsible for carbon dioxide fixation in C4 and crassulacean acid metabolism plants, has also been found to play an important role in carbon metabolism in legume root nodule. PEPC-mediated CO2 fixation in nodules results in the synthesis of C4 dicarboxylic acids, viz. aspartate, malate, fumarate etc. which can be transported into bacteroids with the intervention of dicarboxylate transporter (DCT) protein. PEPC has been purified from the root nodules of few legume species. Information on the relationship between nitrogen fixation and carbon metabolism through PEPC in leguminous plants is scanty and incoherent. This review summarizes the various aspects of carbon and nitrogen metabolism in legume root nodules.     

Metabolic engineering of the malonyl-CoA pathway to efficiently produce malonate in Saccharomyces cerevisiae

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation.     

Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules.

Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.     

Rhizobium trifolii mutants deficient in exopolysaccharide production

Spontaneous mutants of Rhizobium trifolii 24AR5 which did not produce exopoly-saccharide were isolated. The non-mucoid mutants formed small white and ineffective nodules on both red and white clover. These nodules contained infection threads, but only a small number of bacteria were released into nodule cells, and bacteroids were rarely observed. The non-mucoid phenotype was not complemented by the symbiotic plasmid (pJB5JI) of Rhizobium leguminosarum.     

Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.     

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.