Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) fromArabidopsis thaliana

Summary Cysteme synthase, the key enzyme for fixation of inorganic sulfide, catalyses the formation of cysteine from O-acetylserine and inorganic sulfide. Here we report the cloning of cDNAs encoding cysteine synthase isoforms fromArabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial and a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS-B; CSase B), respectively.AtCS-C andAtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (45.8 kD) and of 392 amino acids ( 41.8 kD), respectively. The deduced amino acid sequences of the mitochondrial and plastidic isoforms exhibit high homology even with respect to the presequences. The predicted presequence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed thatAtCS-C is higher expressed in roots than in leaves whereas the expression ofAtCS-B is stronger in leaves. Furthermore, gene expression of both genes was enhanced by sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of theAtCS-B gene is regulated by light. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) isoforms of cysteine synthase ofArabidopsis are able to complement a cysteine synthasedeficient mutant ofEscherichia coli unable to grow on minimal medium without cysteine, indicating synthesis of functional plant proteins in the bacterium. Two lines of evidence proved thatAtCS-C encodes a mitochondrial form of cysteine synthase; first, import ofin vitro translation products derived from AtCS-C in isolated intact mitochondria and second, Western blot analysis of mitochondria isolated from transgenic tobacco plants expressing AtCS-C cDNA/c-myc DNA fusion protein.Abbreviations CSase cysteine synthase The nucleotide sequence data reported will appear in the EMBL Database under the accession numbers X81973 forAtCS-C and X81698 forAtCS-B.     

Spinach pyruvate kinase isoforms : partial purification and regulatory properties

Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K_m values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K_i for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K_a of 0.05 millimolar, and glutamate is an inhibitor with a K_i of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, we suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.     

Purification and properties of cysteine synthase from Spinacia oleracea

Cysteine synthase was purified 3200-fold from Spinacia oleracea leaves. The purified enzyme has an apparent M, of 60 000 ± 2000 and can be dissociated into identical subunits of M, 32 000 ± 2000. The subunits contain one molecule of pyridoxal 5′-phosphate. The K_m value is 2.9 mM for O-acetyl-L-serine and 22 μM for sulphide. Cysteine synthase from S. oleracea catalysed the formation of β-(pyrazol-1-yl)-L-alanine, and β-(3-amino-1,2,4-triazol-1-yl)-L-alanine, and significant differences were found between this enzyme and β-substituted alanine synthases and cysteine synthase from other sources. Amino acid composition of the purified enzyme was also determined.     

Alteration of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase activase activities by site-directed mutagenesis

Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes.     

Major differences in isoform composition of starch synthase between leaves and embryos of pea (Pisum sativum L.)

Isoforms of starch synthase (EC 2.4.1.21) in pea (Pisum sativum L.) leaves have been identified and compared with those in developing pea embryos. Purification and immunoprecipitation experiments show that most of the soluble starch synthase activity of the leaf is contributed by a novel isoform (SSIII) that is antigenically related to the major soluble isoform of the potato tuber. The major soluble isoform of the embryo (SSII) is also present in the leaf, but contributes only 15% of the soluble activity. Study of the leaf starch of lam mutant peas, which lack the abundant granule-bound isoform responsible for amylose synthesis in the embryo (GBSSI), indicates that GBSSI is not responsible for the synthesis of amylose-like material in the leaf. Leaves appear to contain a novel granule-bound isoform, antigenically related to GBSSI. The implications of the results for understanding of the role of isoforms of starch synthase are discussed. Received: 13 March 1997 / Accepted: 13 May 1997     

Purification of nitrate reductase from spinach (Spinacea oleracea L.) by immunoaffinity chromatography using a monoclonal antibody

NADH-Nitrate reductase (EC 1.6.6.1) from spinach (Spinacea oleracea L. v. Noorman) has been purified to apparent homogeneity by immunoaffinity chromatography using a monoclonal antibody linked covalently to Sepharose 4B followed by affinity chromatography. A pre-column of covalently linked non-immune rat γ globulin prevented non-specific binding. The enzyme, released with 1 M KNO₃, was purified 1550-fold to a specific activity of 24.8 μmol NO₂⁻ produced min⁻¹, mg protein⁻¹ with a recovery of 60% of applied NADH-NR activity. Proteolytically ‘nicked’ subunits, detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were removed by 5′-AMP Sepharose chromatography (Fido and Notton, Plant Sci. Lett., 37 (1984) 87).     

Sources of reducing power for nitrate reduction in spinach leaves

The possible source of NADH, the energy donor for nitrate reductase (EC 1.6.6.1), has been studied using an in vivo assay involving freezing the material (leaves of Spinacea oleracea L.) in liquid nitrogen in order to render the tissue permeable to added substrates. Glycolysis and the pentose phosphate pathway were capable of generating NADH through glyceraldehyde-3-phosphate dehydrogenase. Malate and isocitrate were also capable of generating NADH white other organic acids tested were not, including glycolate which was ineffective even under anaerobic conditions.     

Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Serine acetyltransferase (SATase) (EC 2.3.1.30 [EC] ) catalyzes theformation of Oacetyl-L-serine (OAS) from L-serine in the presenceof acetyl-CoA. A novel assay method was developed for measuringthis enzyme activity in extracts from plant tissues. The assayconsists of a coupled system in which the OAS formed is convertedto cysteine by the addition of cysteine synthase (CSase) (EC4.2.99.8 [EC] ). Cysteine thus formed is determined colorimetricallyand serves as a measure for SATase activity. This method israpid, simple and sensitive, and can be readily adapted formeasurement of SATase activity in crude tissue extracts or homogenates. (Received January 14, 1987; Accepted April 27, 1987)     

Glutamine synthetase and glutamate dehydrogenase isoforms in maize leaves: localization, relative proportion and their role in ammonium assimilation or nitrogen transport

 Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C₄ plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed. Received: 25 January 2000 / Accepted: 30 March 2000     

Expression of the rice cytoplasmic cysteine synthase gene in tobacco reduces ozone-induced damage

Cysteine serves as a precursor for the synthesis of various sulfur-containing metabolites, and the cysteine synthase (CS) gene plays a central role in the sulfur cycle in nature. In the present study, rcs1, a cytosolic CS gene of rice, was introduced into the genome of tobacco (Nicotiana tabacum). The tolerance of wild-type tobacco plants as well as of the resulting transgenic tobacco plants overexpressing the rcs1 gene to toxic levels of ozone (O₃, 0.15 μ mol⁻¹) was measured after various lengths of exposure. Leaf lesions in plants exposed for 2 weeks to O₃ were more prevalent in the leaves of the wild-type plants than in those of the transgenic tobacco plants. Transgenic tobacco plants showed a higher growth rate and a higher chlorophyll content than the wild-type plants. Cysteine synthase activity and cysteine and glutathione contents were higher in transgenic plants than in wild-type plants irrespective of the length of the O₃ treatment. Our results indicate that the CS gene plays a role in the protection of the plant against toxic O₃ gas, probably through the mechanism of an over-accumulation of such sulfur-rich antioxidants as cysteine and glutathione.     

Molecular cloning and expression of key enzymes for biosynthesis of cysteine and related secondary non-protein amino acids

Cysteine synthase plays a key role in the sulfur assimilation pathway in plant cells. The cDNA clones encoding two isoforms of this enzyme were isolated from spinach by synthetic oligonucleotide probes. The modes of expression of these two genes differed in tissues of spinach. A heterologous expression system in Escherichia coli and transgenic tobacco was made. The application of heterologous expression to modify sulfur metabolism and to produce non-protein amino acids is discussed.Abbreviation CSase cysteine synthase     

Changes in the Activity of the Chloroplastic and Cytosolic Forms of Dihydroxyacetone Phosphate Reductase during Maturation of Leaves

Young or mature rosette leaves from spinach (Spinacia oleracea L.) plants growing in the field, in the greenhouse, or in a growth chamber under a regimen of 8 hours light and 16 hours dark contained 15 to 50 nanomoles per minute per gram wet weight of NADH:dihydroxyacetone phosphate reductase activity. Of this activity, 75 to 87% was the chloroplastic isoform and 25 to 13% was the cytosolic form. When plants were induced to senesce, as measured by stem elongation and flowering, the percentage of the two reductase isoforms in rosette or stem leaves changed to about 12% as the chloroplastic and 88% as the cytosolic isoform. The change in enzyme activity of the rosette leaves occurred within 3 days, before phenotypic changes were observed. Likewise, when plants senesced in continuous darkness, the percentage of chloroplastic to cytosolic reductase changed from 80:20% to 25:75% after 62 hours before changes in total protein or chlorophyll occurred. The ratio of activities did not change in the first 16 hours of darkness or overnight. In each case the change in ratio resulted from about a 75% decrease in activity of the chloroplastic isoform and up to 14-fold increase in cytosolic isoform. In spinach leaves purchased at a local market primarily only the cytosolic isoform remained. When plants were returned to normal day-nights, after 62 hours in continuous darkness, the activity of the chloroplastic isoform increased, but not to control levels after 3 days, while the cytosolic enzyme decreased within 1 day to normal day-night values. Changes in activity were not due to changes during in vitro assays in activation by thioredoxin for the chloroplastic isoform or fructose 2,6-phosphate for the cytosolic isoform.     

Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.)

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.     

Ornithine Carbamoyltransferase from Spinacea oleracea: Purification and Characterization

Ornithine carbamoyltransferase (OCT) from spinach (Spinacea oleracea L.) was purified to homogeneity and studied for some kinetic and structural properties. The enzyme showed a specific activity of 436 U mg^–1, its molecular mass was approximately 118 kDa as estimated by Sephacryl S-200 gel filtration chromatography, the purified protein ran as a single band of 38 kDa in sodium dodecyl sulfate-polyacryamide gel electrophoresis. The enzyme catalyses an ordered bi-bi-sequential reaction in which carbamoyl phosphate binds first, followed by L-ornithine; L-citrulline leaves first, followed by phosphate. The Michaelis constant was 0.19 mM for L-ornithine and 13.1 µM for carbamoyl phosphate; the dissociation constant for the enzyme and carbamoyl phosphate complex was of 19 µM. The K_m of the reaction decreases from pH 6.0 to pH 10.4. The enzyme is heat-labile, but it was protected from thermal inactivation by substrates; more by ornithine alone than by two substrates acting together.     

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine synthase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.     

Activities of Cysteine Synthetase and Cystathionase in Bacilhis subtilis during Sporulation

Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A–50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.

The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores.     

Purification and characterization of thermostable cellulase from Enterobacter cloacae IP8 isolated from decayed plant leaf litter

Cellulases are important in the hydrolysis of lignocellulosic materials and thereby contribute to biomass conversion into fuels and chemicals. A cellulase-producing bacterium was isolated from decayed plant leaf litter in soil of a botanical garden. Based on morphological, biochemical and 16S rRNA gene sequencing, it was identified as Enterobacter cloacae IP8, with gene bank accession number NR118568.1. The bacterial cellulase was purified in a three-step procedure using lyophilization, ion exchange chromatography (QAE Sephadex A-50) and gel filtration (Biogel P-100). Two isoforms of the enzyme were purified 1.21 and 1.23 folds, respectively, with yields of 30 and 29% for isoforms A and B, respectively. Apparent molecular weights of 36.61?±?1.40 and 14.1?±?0.10?kDa were obtained for isoforms A and B, respectively, using gel filtration chromatography. Kinetic parameters K_m and V_max were 0.13?±?0.04?mg/ml and 3.84?±?0.05?U/ml/min, respectively, for isoform A and 0.58?±?0.06?mg/ml and 13.8?±?0.10?U/ml/min, respectively, for isoform B. Optimum pH (7.0) and temperature (60?°C) of cellulase activity were determined for both isoforms A and B. Na⁺ and Ca²⁺ enhanced the activities of both isoforms. Mg²⁺ inhibited the enzyme activity at concentrations 4–15?mM but, while it stimulated the activity of isoform A at concentrations 15–200?mM, it inhibited that of isoform B at same concentration range. The strong inhibition of the enzyme by ethylenediaminetetraacetic acid (EDTA) confirmed the enzyme as a metalloenzyme. These results reveal the purified cellulase from E. cloacae IP8 as a thermostable, acidic to neutral metalloenzyme, suggesting that it has good potential for biotechnological applications.     

Biochemical and molecular characterization of a novel starch synthase from potato tubers

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N-terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule-bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity. The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.