Functional analysis and regulation of the malate synthase from<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Malate synthase (EC 2.3.3.9, formerly EC 4.1.2.2) has been investigated in the unicellular green algae Chlamydomonas reinhardtii. The molecular characteristics and the regulation of gene expression have been investigated for the enzyme. A full-length malate synthase cDNA has been isolated, containing an open reading frame of 1,641 bp encoding a polypeptide of 546 amino acids. This protein shares the conserved signature of the malate synthase family, along with the catalytic residues essential for enzymatic activity and a C-terminal motif that matches the consensus for glyoxysome import. Functionality studies have been facilitated by heterologous expression of the malate synthase cDNA in Escherichia coli. The remarkable metabolic versatility of the alga has been used to analyse the metabolic control of malate synthase gene expression. The data strongly support the role of acetate and light as the main regulatory effectors, and the existence of cross-talk between the two signalling pathways.Abbreviations IPTG Isopropyl -d-thiogalactopyranoside - MS Malate synthase - PCR Polymerase chain reaction - PTS Peroxisomal targeting sequence - RACE Rapid amplification of cDNA ends - TAP Tris–acetate–phosphate medium - TCA Tricarboxylic acid cycle     

Identification and disruptional analysis of the Streptomyces cinnamonensis msdA gene, encoding methylmalonic acid semialdehyde dehydrogenase

The msdA gene encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is known to be involved in valine catabolism in Streptomyces coelicolor. Using degenerative primers, a homolog of msdA gene was cloned and sequenced from the monensin producer, Streptomyces cinnamonensis. RT-PCR results showed msdA was expressed in a vegetative culture, bump-seed culture and the early stages of oil-based monensin fermentation. However, isotopic labeling of monensin A by [2, 4-¹³C₂]butyrate revealed that this MSDH does not play a role in providing precursors such as methylmalonyl-CoA for the monensin biosynthesis under these fermentation conditions. Using a PCR-targeting method, msdA was disrupted by insertion of an apramycin resistance gene in S. cinnamonensis C730.1. Fermentation results revealed that the resulting ΔmsdA mutant (CXL1.1) produced comparable levels of monensin to that observed for C730.1. This result is consistent with the hypothesis that butyrate metabolism in S. cinnamonensis in the oil-based fermentation is not mediated by msdA, and that methylmalonyl-CoA is probably produced through direct oxidation of the pro-S methyl group of isobutyryl-CoA. The CXL1.1 mutant and C730.1 were both able to grow in minimal medium with valine or butyrate as the sole carbon source, contrasting previous observations for S. coelicolor which demonstrated msdA is required for growth on valine. In conclusion, loss of the S. cinnamonensis msdA neither affects valine catabolism in a minimal medium, nor butyrate metabolism in an oil-based medium, and its role remains an enigma.     

Thermostable malate synthase of<Emphasis Type="Italic"> Streptomyces thermovulgaris</Emphasis>

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40–60 °C) than those of scMS (28–37 °C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9–91.4% amino acid identities, with stMS possessing slightly more charged residues (~31%) than its mesophilic counterparts (~28–29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

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.     

Evidence for the presence of the glyoxylate cycle in Chloroflexus

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present in cell-free extracts of the phototrophic, green, thermophilic bacterium Chloroflexus aurantiacus grown with acetate as the sole organic carbon source.The optimum temperature of these enzymes was 40° C, and their specific activities were high enough to account for the observed growth rate. Lower levels of the enzymes were found in extracts from cells grown on a complete medium.Itaconate was shown to inhibit isocitrate lyase from C. aurantiacus 96% at a concentration of 0.25 mM and also had a profound effect on the growth of the organism on acetate, 0.25 mM inhibiting completely. Itaconate also inhibited the growth when added to the complex medium, but in this case much higher concentrations were required.     

Acetate thiokinase and the assimilation of acetate in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum growing on H₂ plus CO₂ as sole carbon and energy source was found to contain acetate thiokinase (Acetyl CoA synthetase; EC 6.2.1.1): Acetate+ATP+CoA Acetyl CoA+AMP+PPi. The apparent K _m value for acetate was 40 M. Acetate kinase (EC 2.7.2.1) and phosphotransacetylase (EC 2.3.1.8) could not be detected. The specific activity of acetate thiokinase was high in cells grown with limited H₂ and CO₂ supply (approximately 100nmol/min · mg protein), it was low in exponentially grown cells (2 nmol/min·mg protein). This corresponded with the finding that cells growing linearly in the presence of acetate assimilated the monocarboxylic acid in high amounts (>10% of the cell carbon was derived from acetate), whereas exponentially growing cells did not (<1% of cell carbon was derived from acetate). These latter observations indicated that acetate thiokinase and free acetate are not involved in autotrophic CO₂ fixation in M. thermoautotrophicum. The presence and some kinetic properties of succinate thiokinase (EC 6.2.1.5), adenylate kinase (EC 2.7.4.3), and inorganic pyrophosphatase (EC 3.6.1.1.) are also described.     

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min^-1·mg^-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min^-1·mg^-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a R_f value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg²⁺ and cysteine.Abbreviations TCA Tricarboxylic acid - ICL isocitrate lyase - MS malate synthase - FPLC fast protein liquid chromatography - maximum specific oxygen consumption rate     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Chloroflexus aurantiacus secretes 3-hydroxypropionate,a possible intermediate in the assimilation of CO2 and acetate

Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 M). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO₂ fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using ¹³C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO₂. The 3HP formed by cells grown on propionate and ¹³CO₂ was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO₂ fixation.Abbreviations 3HP 3-hydroxypropionate - 2F3HP 2,fluoro,3-hydroxypropionate - FAc fluoroacetate - GC gas chromatography - MS mass spectrometry - NMR nuclear magnetic resonance     

Induction of glyoxylate cycle enzymes in rat liver upon food starvation

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The K_m(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a K_m(acetyl-CoA) of 0.2 μM, a K_m(glyoxylate) of 3 mM and a pH optimum of 7.6.     

Taurine catabolism: III. Evidence for the participation of the glyoxylate cycle

The metabolic fate of acetate, produced during taurine catabolism in Pseudomonas aeruginosa TAU-5, appear to involve the glyoxylate cycle. Organisms grown on taurine have significantly higher levels of malate synthetase and isocitrate lyase than cells grown on nutrient broth, but were comparable to the levels found in acetate-grown organisms. Itaconate, an isocitrate lyase inhibitor, produced a prolonged lag phase and reduced the growth rate of organisms when it was present in the taurine or acetate growth medium. Ethylmethanesulfonate treatment of TAU-5 yielded mutant strains unable to grow on taurine or acetate as sole carbon sources, due to a lack of either malate synthetase or isocitrate lyase. Spontaneous revertants derived from these mutant strains regained the missing enzyme activity and the ability to grow on taurine or acetate.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

Occurrence and evolutionary significance of two sulfate assimilation pathways in the rhodospirillaceae

The ability to use adenosine 5-phosphosulfate (APS) or 3-phosphoadenosine 5-phosphosulfate (PAPS) as the substrate for the initial reductive step in sulfate assimilation has been tested in most of the known Rhodospirillaceae species and in some chemotrophic bacteria. Improved and optimized methods for the synthesis and purification of the sulfonucleotides APS and PAPS are described. The production of acid volatile radioactivity from ³⁵S-APS and ³⁵S-PAPS was measured under various conditions in the presence and absence of non-labeled sulfate. Specific differences in the ability to reduce APS or PAPS were observed among the Rhodospirillaceae species and also the chemotrophic bacteria. APS was found to be the substrate of the thiolsulfotransferase in Rps. acidophila, Rps. globiformis, Rm. vannielii, Rc. purpureus, R. tenue, Rps. gelatinosa, in Alcaligenes eutrophus and Pseudomonas aeruginosa. PAPS was the substrate in Rps. capsulata, Rps. sphaeroides, Rps. sulfidophila, Rps. palustris, Rps. viridis, R. rubrum, R. fulvum, in Paracoccus denitrificans and in several Enterobacteriaceae. The presence of different enzymatic systems for sulfate reduction in the Rhodospirillaceae family is compared with their taxonomical grouping and their possible phylogenetic relatedness.Nonstandard Abbreviations APS adenosine 5-phosphosulfate - PAPS 3-phosphate adenosine 5-phosphosulfate - DTE dithioerythrol - Rc. Rhodocyclus - R. Rhodospirillum - Rm. Rhodomicrobium - Rps. Rhodopseudomonas     

Characterization of a malate dehydrogenase in the cyanobacterium Coccochloris peniocystis

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a K_m (malate) of 5.6 mM and K_ms for NAD and NADP of 24 M and 178 M, respectively, although similar V_max were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly ¹⁴C-labelled malate caused no ¹⁴CO₂ release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.Abbreviations MDH malate dehydrogenase - PEPcase phosphoenolpyruvate carboxylase - MOPS 3-[N-Morpholino] propane sulfonic acid - TRIS Tris(hydroxymethyl)-aminomethane - EDTA Disodium Ethylenadiamine Tetraacetate - MES 2[N-Morpholino]-ethane Sulfonic Acid - EPPS N-2-Hydroxyethylpiperazine Propane - MW Molecular weight - OAA Oxaloacetic acid     

Relationship between nitrogen assimilation and cephalosporin synthesis inStreptomyces clavuligerus

The levels of three enzymes of the -lactam antibiotic pathway and overall cephalosporin production were subject to nitrogen source repression inStreptomyces clavuligerus. The specific activities of isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) measured during the exponential phase depended on the nitrogen source employed, following a pattern that roughly correlated with the corresponding antibiotic production. The effects on isopenicillin N epimerase (epimerase) activities were less marked than those on the cyclase and expandase.Production of cephalosporins and enzymatic activities were not related to the growth rate of the cultures. Glutamate, glutamine and alanine inhibited production when added to resting cell systems, while lysine and -aminoadipate were stimulatory. No clear relationship could be drawn between cephalosporin production or -lactam synthetase activities and the activities of enzymes of ammonium assimilation (glutamine synthetase, glutamate synthase and alanine dehydrogenase).The intracellular pools of free glutamine, alanine and ammonium were the only ones markedly affected by the nitrogen source in the wild type and mutants, but these amino acids did not seem to play an obvious role as intracellular mediators of nitrogen control.Abbreviations DCW dry cell weight - GS glutamine synthetase - GOGAT glutamate synthase - ADH alanine dehydrogenase - HPLC high performance liquid chromatography     

Heterotrophic nitrification by Arthrobacter sp. (strain 9006) as influenced by different cultural conditions,growth state and acetate metabolism

An Arthrobacter sp. (strain 9006), isolated from lake water, accumulated nitrite up to about 15 mg N/l, but no nitrate. In a mineral medium supplemented with tryptone, yeast extract, acetate and ammonium, the cells released nitrite into the medium parallel to growth or when growth had virtually ceased. The nitrite formed was proportional to the initial acetate concentration, indicating an involvement of acetate metabolism with nitrification. The organism grew with a wide variety of organic carbon sources, but washed cells formed nitrite from ammonium only in the presence of citrate, malate, acetate or ethanol. Magnesium ions were required for nitrification of ammonium and could not be replaced by other divalent metal ions. Analysis of the glyoxylate cycle key enzymes in washed suspensions incubated in a minimal medium revealed that isocitrate lyase and malate synthase were most active during the nitrification phase. Nitrite accumulation but not growth was inhibited by glucose, tryptone and yeast extract. A possible explanation for the different nitrification patterns during growth is based on the regulatory properties of glyoxylate cycle enzymes.Abbreviations IL Isocitrate lyase [threo-D_s-isocitrate glyoxylate-lase, E.C. 4.1.3.1.] - MS malate synthase [l-malate glyoxylate-lyase (CoA-acetylating), E.C. 4.1.3.2.]     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.