PCR-mediated screening and cloning of a malate synthase genefrom Streptomyces griseus NCIMB 9001

Malate synthase is an essential metabolic enzyme of the glyoxylate bypass that makes possible the replenishment of carbon intermediates to cells grown on acetate. A polymerase chain reaction (PCR)-based molecular screening investigation of full-length malate synthase genes from Streptomyces spp. was initiated by our group. To this end, consensus primers were designed based on known streptomycete malate synthase sequences and successful amplification was obtained for Streptomyces griseus, S. fimbriatus and S. lipmanii. The putative full-length malate synthase gene from S. griseus was subsequently cloned, sequenced and expressed. Sequence analysis of this gene showed very high identity with other streptomycete malate synthase genes. Furthermore, high malate synthase activity was detected after heterologous expression in Escherichia coli, thus demonstrating successfully the rapid cloning and functional verification of a streptomycete malate synthase gene. Growth studies of S. griseus revealed that malate synthase activity was induced by the presence of acetate, which is a two-carbon source. Interestingly, the activity peaked during late growth phase when the biomass was declining, suggesting that the enzyme may have a late role in metabolism.     

Kinetics of malate transport and decomposition in acid soils and isolated bacterial populations: The effect of microorganisms on root exudation of malate under Al stress

The kinetics and characteristics of malate degradation were studied in four acid soils ranging in both pH (4.30 to 5.00) and vegetation type. The breakdown of malate was rapid in all soils with a half life of approximately 1.7 h, K_m of 1.7 mM and V_max of 70 nmol g^–1 soil h^–1. No relationship was observed between malate decomposition rate and pH. Co-metabolism studies with other C and N substrates (glucose, glycine, glutamate, citrate and succinate) indicated that the microorganisms were not N limited and competitive inhibition of malate breakdown was only observed in the presence of succinate. Studies with isolated mixed bacterial cultures indicated that the bacterial malate uptake was mediated by an energy dependent, dicarboxylate transporter which can be inhibited by succinate and is independent of pH between pH 5.0 and 7.0. The K_m and V_max parameters ranged from 279–955 M and 0.1–17 mol mg^–1 protein h^–1 for the mixed bacterial cultures depending on the bacteria's previous C source. The results indicate that in acid topsoils where microbial populations are high, the microbes may provide a considerable sink for organic acids. If organic acids are being released by roots in response to an environmental stress (e.g. Al toxicity, P deficiency) it can be expected that the efficiency of these root mediated metal resistance mechanisms will be markedly reduced by rapid microbial degradation.     

Direct transfer of NADH from malate dehydrogenase to Complex I in Escherichia coli

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH: ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate+NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate+NAD was estimated to be 1 μM, while the rate of proton translocation due to Complex I was consistent with a some what higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity. These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.     

Brassinosteroid signaling regulates phosphate starvation-induced malate secretion in plants

Inorganic phosphate (Pi) is often limited in soils due to precipitation with iron (Fe) and aluminum (Al). To scavenge heterogeneously distributed phosphorus (P) resources, plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe-P or Al-P oxides. In this study, we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE-RESISTANT 1 (BZR1) expression in Arabidopsis thaliana. Exogenous 2,4-epibrassinolide treatment or constitutive activation of BZR1 (in the bzr1-D mutant) significantly reduced primary root growth inhibition under Pi-starvation conditions by downregulating ALUMINUM-ACTIVATED MALATE TRANSPORTER 1 (ALMT1) expression and malate secretion. Furthermore, AtBZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1 (STOP1) on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis. The ratio of nuclear-localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis. In addition, BZR1-inhibited malate secretion is conserved in rice (Oryza sativa). Our findings provide insight into plant mechanisms for optimizing the secretion of malate, an important carbon resource, to adapt to Pi-deficiency stress.     

Isolation and characterization of an apple cytosolic malate dehydrogenase gene reveal its function in malate synthesis

Cytosolic NAD-dependent malate dehydrogenase (cyMDH) is an enzyme crucial for malate synthesis in the cytosol. The apple MdcyMDH gene (GenBank Accession No. DQ221207) encoding the cyMDH enzyme in apple was cloned and functionally characterized. The protein was subcellularly localized to the cytoplasm and plasma membrane. Based on kinetic parameters, it mainly catalyzes the reaction from oxalacetic acid (OAA) to malate in vitro. The expression level of MdcyMDH was positively correlated with malate dehydrogenase (MDH) activity throughout fruit development, but not with malate content, especially in the ripening apple fruit. MdcyMDH overexpression contributed to malate accumulation in the apple callus and tomato. Taken together, our results support the involvement of MdcyMDH directly in malate synthesis and indirectly in malate accumulation through the regulation of genes/enzymes associated with malate degradation and transportation, gluconeogenesis and the tricarboxylic acid cycle.     

Enhanced exudation of malate in the rhizosphere due to AtALMT1 overexpression in blackgram (Vigna mungo L.) confers increased aluminium tolerance

• Worldwide, 50% of soil is acidic, which induces aluminium (Al) toxicity in plants, as the phyto‐availability of Al³⁺ increases in acidic soil. Plants responds to Al³⁺ toxicity by exuding organic acids into the rhizosphere. The organic acid responsible for Al³⁺ stress response varies from species to species, which in the case of blackgram (Vigna mungo L.) is citrate.
• In blackgram, an Arabidopsis malate transporter, AtALMT1, was overexpressed with the motive of inducing enhanced exudation of malate. Transgenics were generated using cotyledon node explants through Agrobacterium tumefaciens‐mediated transformation. The putative transgenics were initially screened by AtALMT1‐specific genomic DNA PCR, followed by quantitative PCR. Two independent transgenic events were identified and functionally characterized in the T₃ generation.
• The transgenic lines, Line 1 and 2, showed better root growth, relative water content and chlorophyll content under Al³⁺ stress. Both lines also accounted for less oxidative damage, due to reduced accumulation of ROS molecules. Photosynthetic efficiency, as measured in terms of F_v/F_m, NPQ and Y(II), increased when compared to the wild type (WT). Relative expression of genes (VmSTOP1, VmALS3, VmMATE) responsible for Al³⁺ stress response in blackgram showed that overexpression of a malate transporter did not have any effect on their expression. Malate exudation increased whereas citrate exudation did not show any divergence from the WT. A pot stress assay found that the transgenics showed better adaptation to acidic soil.
• This report demonstrates that the overexpression of a malate transporter in a non‐malate exuding species improves adaptation to Al³⁺ toxicity in acidic soil without effecting its stress response mechanism.

     

Vacuolar malate uptake is mediated by an anion-selective inward rectifier

Electrophysiological studies using the patch‐clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current–voltage relations of the tonoplast were dominated by a strongly inward‐rectifying anion‐selective channel that was active at cytoplasmic‐side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage‐dependent gating of a 3‐pS malate‐selective ion channel, which showed a half‐maximal open probability at ?43 mV. Also, the time‐averaged unitary currents following a step to a negative voltage corresponded to the time‐dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion‐selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent K_m of 2.5 mm malate^2? activity), a selectivity sequence of fumarate^2? > malate^2? > Cl^? > maleate^2– ≈ citrate^3–, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 µm niflumate reversibly blocked the activity of the 3‐pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 µm^?2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3‐pS, inward‐rectifying, anion‐selective channel represents the principal pathway for malate influx into the vacuole.     

Redirection of the phenylpropanoid pathway to feruloyl malate in <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants deficient for cinnamoyl-CoA reductase 1

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25–35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.     

Deciphering functional redundancy and energetics of malate oxidation in mycobacteria

Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.     

On the consequences of aluminium stress in rye: repression of two mitochondrial malate dehydrogenase mRNAs

Plants have developed several external and internal aluminium (Al) tolerance mechanisms. The external mechanism best characterised is the exudation of organic acids induced by Al. Rye (Secale cereale L.), one of the most Al‐tolerant cereal crops, secretes both citrate and malate from its roots in response to Al. However, the role of malate dehydrogenase (MDH) genes in Al‐induced stress has not been studied in rye. We have isolated the ScMDH1 and ScMDH2 genes, encoding two different mitochondrial MDH isozymes, in three Al‐tolerant rye cultivars (Ailés, Imperial and Petkus) and one sensitive inbred rye line (Riodeva). These genes, which have seven exons and six introns, were located on the 1R (ScMDH1) and 3RL (ScMDH2) chromosomes. Exon 1 of ScMDH1 and exon 7 of ScMDH2 were the most variable among the different ryes. The hypothetical proteins encoded by these genes were classified as putative mitochondrial MDH isoforms. The phylogenetic relationships obtained using both cDNA and protein sequences indicated that the ScMDH1 and ScMDH2 proteins are orthologous to mitochondrial MDH1 and MDH2 proteins of different Poaceae species. The expression studies of the ScMDH1 and ScMDH2 genes indicate that it is more intense in roots than in leaves. Moreover, the amount of their corresponding mRNAs in roots from plants treated and not treated with Al was higher in the tolerant cultivar Petkus than in the sensitive inbred line Riodeva. In addition, ScMDH1 and ScMDH2 mRNA levels decreased in response to Al stress (repressive behaviour) in the roots of both the tolerant Petkus and the sensitive line Riodeva.     

Functioning of malate dehydrogenase system in mesophyll and bundle sheath cells of maize leaves under salt stress conditions

The formation of adaptive response to salt stress in mesophyll and bundle sheath cells of maize (Zea mays L.) leaves was studied at the level of operation of enzyme systems that participate in oxidation of malate. Functioning of four malate dehydrogenases (MDH), the components of this system, was studied and found to maintain malate and pyruvate pools, which are required for operation of the Hatch-Slack cycle and actively used for neutralization of salt treatment. The increase in activity of NAD-MDH was related to salt-induced synthesis of the additional isoform of MDH in mesophyll cells. Such changes in the isozyme pattern were not found in bundle sheath cells.     

Electrophoretic variation of supernatant malate dehydrogenase in marsupials

Starch gel electrophoresis of supernatant malate dehydrogenase (MDH A₂) was performed on erythrocyte samples from 505 individual animals representative of 33 marsupial species. Most species exhibited electrophoretically identical forms of MDH A₂ activity with the exception of the grey kangaroos, Trichosurus possums, and bandicoots, thus confirming the phylogenetic relatedness of animals within each group and the conservative nature of this enzyme. Polymorphisms were observed in two of the six species analyzed whose mobilities were non-standard. Allelic isozyme patterns and those from interspecies F₁ hybrids between grey kangaroos and other macropods were consistent with a dimeric subunit structure and an autosomal locus (MDH-A) encoding the enzyme.Supported in part by grants from the Australian Research Grants Committee.     

Seasonal pattern of mannitol and malate accumulation in leaves of two manna ash species (Fraxinus ornus L. and F. angustifolia Vahl) growing in Sicily

ABSTRACT

The content of mannitol and malate was assayed enzimatically during spring, summer and autumn, in leaves of two species of ash, Fraxinus ornus L. and Fraxinus angustifolia Vahl, traditionally cultivated in Sicily for the extraction of manna. Both species contain high levels of mannitol and show, on a dry weight basis, a 65–80% increase in the summer content of this polyol. The malate content differs in the two species: in F. ornus it shows a summer increase, but it is relatively low (65–115 µmol g^-1 DW), while in F. angustifolia it is higher (275–318 µmol g^-1 DW), but remains more or less constant throughout the year. The results suggest that in these species, under the local field conditions, mannitol has a more relevant role than malate in the response to summer drought.     

Effect of nitrate limitation on the photosynthetically active pools of aspartate and malate in maize, a NADP malic enzyme C4 plant

After two weeks of moderate N restriction, growth of 3-week-old Zea mays L. plants was less than half that of the control and aspartate and malate levels in the leaves were severely suppressed (45 and 65% decrease, respectively). Since in NADP malic enzyme type C₄ plants, such as maize, malate and aspartate are intermediates in the C₄ photosynthetic pathway, the operation of the latter was investigated. Moderate nitrogen deficiency had only a small effect on the rate of photosynthesis (20% decrease) measured under 1000 umol m^?2 s^?1 irradiance. ¹⁴CO₂ pulse-¹²CO₂ chase experiments combined with measurements of in vitro photosynthetic enzyme activities demonstrated the operation of a typical C⁴ photosynthetic pathway in N-restricted plants. The turnover rates of malate and aspartate molecules involved in the C₄ cycle were determined by the loss of label in the carbon 4 moiety of these molecules during the chase period. It is shown that N restriction did not alter the turnover of malate but greatly accelerated that of aspartate. The amounts of malate and aspartate moving through photosynthetically active pools were estimated using a kinetic model. For malate, the size of this pool appeared to be only slightly diminished whereas for aspartate the size of the corresponding pool decreased by a factor of 3. It is proposed that under moderate NO₃^? deficiency, despite deviations in malate metabolism leading to a pronounced decrease in the size of its cellular pool, a large amount of malate remained in the operation of the C₄ pathway. By contrast, the participation of aspartate in the operation of the C₄ pathway was greatly reduced.     

Several lanthanides activate malate efflux from roots of aluminium‐tolerant wheat

Many elements of the lanthanide series exist as trivalent cations in solution below pH 6. The present study was carried out to investigate whether lanthanides could stimulate malate efflux from wheat (Triticum aestivum L.) roots, as has been found for trivalent aluminium (Al) cations. Excised root apices treated with 100 µm of each of seven different lanthanide elements (lanthanum, praseodymium, europium, gadolinium, terbium, erbium, and ytterbium) stimulated malate efflux, with five‐ to fifty‐fold more malate being released from an Al‐tolerant wheat line than from a near‐isogenic Al‐sensitive line. As erbium stimulated the greatest malate efflux of the lanthanides tested, this response was characterized further. The characteristics of the erbium‐activated efflux were similar to the Al‐activated efflux described previously suggesting that both of these ions activate the same transport mechanism. The capacity for erbium‐activated malate efflux cosegregated with Al tolerance in wheat seedlings derived from a cross between Al‐sensitive and Al‐tolerant near‐isogenic lines. This is the first study to identify cations, other than Al, which can activate malate release from wheat roots. It also provides additional evidence that malate efflux from root apices is the primary mechanism for Al tolerance in wheat.     

Molecular dynamic simulations of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor

The beta-lactam-based antibiotics are among the most prescribed and effective antibacterial agents. Widespread use of these antibiotics, however, has created tremendous pressure for the emergence of resistance mechanisms in bacteria. The most common cause of antibiotic resistance is bacterial production of actamases that efficiently degrade antibiotics. The metallo-beta-lactamases are of particular clinical concern due to their transference between bacterial strains. We used molecular dynamics (MD) simulations to further study the conformational changes that occur due to binding of an inhibitor to the dicanzinc metallo-beta-lactamase from Bacteroides fragilis. Our studies confirm previous findings that the major flap is a major source of plasticity within the active site, therefore its dynamic response should be considered in drug development. However, our results also suggest the need for care in using MD simulations in evaluating loop mobility, both due to relaxation times and to the need to accurately model the zinc active site. Our study also reveals two new robust responses to ligand binding. First, there are specific localized changes in the zinc active site—a local loop flip—due to ligand intercalation that may be critical to the function of this enzyme. Second, inhibitor binding perturbs the dynamics throughout the protein, without otherwise perturbing the enzyme structure. These dynamic perturbations radiate outward from the active site and their existence suggests that long-range communication and dynamics may be important in the activity of this enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of sulphite on phosphoenolpyruvate carboxylase and malate formation in extracts of Zea mays

The effect of SO₃^2? on the activity of PEP-carboxylase and on subsequent malate formation has been studied in leaf extracts of Zea mays. PEP-carboxylase was assayed by incorporation of H¹⁴CO₃ - into oxaloacetate dinitrophenylhydrazone and by a spectrophotometric method. In contrast to ribulose diphosphate carboxylase, PEP-carboxylase was not inhibited by 10 mM SO₃^2? with respect to PEP. As was the case with ribulose diphosphate carboxylase, the activity of PEP-carboxylase was inhibited non-competitively with respect to Mg²⁺. However, the K_i value (84.5 mM) was found to be very high. With respect to HCO₃^?, like ribulose diphosphate carboxylase, PEP-carboxylase was inhibited competitively, but the K_i value (27 mM SO₃^2?) increased by about the same factor (× 9) as the K_m, (0·5 mM HCO₃^?) is decreased. This indicates that the replacement of HCO₃^? by SO₃^2?, common to both enzymes, is facilitated by decreasing the affinity of the enzyme for HCO₃^?. At substrate saturating conditions malate formation by the combined action of PEP-carboxylase and endogenous NADH-dependent malate dehydrogenase in leaf extracts was not inhibited by 10 mM SO₃^2?. Although the malate dehydrogenase is inhibited at this SO₃^2? concentration to about 85%, malate formation is unaffected, as PEP-carboxylase is the rate limiting step its turnover rate being only about 8% of NADH-dependent malate dehydrogenase.     

The malate synthase gene of cucumber

The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined. The sequences have enabled us to identify putative control regions at the 5 end of the gene, three introns, and possible alternative polyadenylation sites at the 3 end. The deduced amino acid sequence predicts a polypeptide of 64961 molecular weight, which has 48% identity with that of Escherichia coli. Comparison of the sequence of malate synthase from cucumber with that from E. coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies.