Biosynthesis of PAPS by algal or higher plant material has occasionally been reported (references... more Biosynthesis of PAPS by algal or higher plant material has occasionally been reported (referencesa in Table 1) and, so far, three different types of enzymatic assays for the APS-kinase have been introduced (referencesb in Table 1). The presence of APS-kinase (EC 2.7.1.25, ATP: adenylylsulfate-3′-phosphotransferase) in plants seems to be ascertained. But, as compared to other enzymes involved in the assimilatory reduction of sulfate, detailed investigations of the APS-kinase are not available.
Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin-depen... more Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin-dependent reduction of 3'-phosphoadenylylsulphate (PAPS) to sulphite. The enzyme became detectable when heterologous thioredoxin from Escherichia coli was used substituting for the hitherto unidentified plant thioredoxin. The enzyme's cross-reactivity with heterologous thioredo xin enabled the partial purification and brief characterization. The molecular weight of the en zyme as estimated by HPLC size exclusion and gel filtration was 68-72 k. The protein reduced PAPS only when thioredoxin was present as cosubstrate. The function of this enzyme in the assimilation of inorganic sulphate by higher plants is discussed in comparison to the function of the respective enzymes from Escherichia coli and Saccharom yces cerevisiae.
Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 m... more Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared "repressed" by the S-amino acids but the GSH-dependent enzyme was "derepressed." In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells.
The structural genes of enterobacteria encoding for the enzymes involved in the assimilatory redu... more The structural genes of enterobacteria encoding for the enzymes involved in the assimilatory reduction of sulphate (“cys genes”) were used in order to identify homologous genes from phototrophic cyanobacteria and higher plants. By Southern hybridisation of genomic DNA from the higher organisms with the cys DNA-probes derived from Escherichia coli, discrete restriction fragments were found in higher plants and in cyanobacteria indicating the occurrence of related DNA. Two of the cyanobacterial genes were cloned and identified by DNA and amino acid sequence comparison as the structural genes encoding the PAPS-reductase (EC 1.8.4.-) and the ferredoxin: sulphite-reductase (EC 1.8.7.1). The nucleic acid of both genes showed stretches of highly conserved bases in regions of the sequences which are regarded as the functionally important domains of the gene products.
Tke common adenine nucleotides (adenosine mono-, di-, triphosphate) have been separated by paired... more Tke common adenine nucleotides (adenosine mono-, di-, triphosphate) have been separated by paired-ion chromatography from tkeir sulpkated derivatives (adenosine S-sulpkatopkospkate, adenosine 3'-phosphate S-sulphatophosphate). With tetrabutylammonium hydroxide as the ion-pair forming reagent, nicotinamideadenine dinucleotide phosphate, Gavin-adenine dinucleotide and adenosine 3'S'-bisphospkate were also separated from these nucleotides by rapid isocratic elution. The method is highly reliable as shown by the capacity factors (I?), and its compatibility with the requirements of a continuous-ff ow radio detection for 3*S-lakelled nucleotides is demonstrated. It has keen apptied to an investigation of tke kinetics of a PAPS sulpkotransferase reaction involved in kigker plant assimilative sulpkate reduction.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
A cDNA clone with an open reading flame of 831 nucleotides was isolated from a AZaplI-library of ... more A cDNA clone with an open reading flame of 831 nucleotides was isolated from a AZaplI-library of Arabidopsis thaliana. The nucleotide sequence of the cDNA is homologous to the APS-kinase genes from enterobacteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53.2%), Rh&obium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14: 57.1%). The polypeptide deduced from the plant APS-kinase cDNA is comprised of 276 amino acid residues with a molecular weight of 29 790. It contains an N-terminal extension of 77 amino acids. This extension includes a putative transit peptide of 37 residues separated from the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26 050 is predicted for the processed protein. The relatedness between bacterial, fungal and plant APS-kinase polypeptides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. meliloti), and 50.3% (Azospirillum brasilense). The plant polypeptide contains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxxGK. Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif described for PAPS dependent enzymes. The identity of the gene was confirmed by analyzing the function of the gene product. The putative transit peptide was deleted by PCR and the truncated gene was expressed in a pTacl vector system. A polypetide of MW 25761 could be induced by IPTG. The gene product was enzymatically active as APS-kinase. It produced PAPS from APS and ATP-the absence of ATP but supplemented with thiols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransferase is not a separate enzyme but identical with APS-kinase.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1973
1. It has been proposed that Mg 2+, inorganic pyrophosphate and a protein fraction which exhibits... more 1. It has been proposed that Mg 2+, inorganic pyrophosphate and a protein fraction which exhibits fructose-l,6-dlphosphatase activity may interact to regulate photosynthesis by isolated chloroplasts. 2. Evidence is presented which confirms the interaction and regulation but shows that these effects are indirectly attributable to pyrophosphatase activity rather than fructose-1,6-dlphosphatase 3. When provided with Mg 2+ and PP, the pyrophosphatase simply alters the proportions of orthophosphate and PP, m the reaction mixture. As the P, concentration is increased, it first stimulates and then mhxblts, the degree of inhiblt~on being enhanced by additional Mg 2 +. PPI ameliorates the inhibition, possibly by chelation of Mg 2 +. 4 It is concluded that the proposed regulation is ultimately governed by the P, concentration and the known relationship between P, uptake and those phosphate export across the chloroplast envelope.
Acta Crystallographica Section D Biological Crystallography, 1998
PAPS reductase from E. coli is involved in sulfur metabolism and catalyses the reduction of phosp... more PAPS reductase from E. coli is involved in sulfur metabolism and catalyses the reduction of phospho-adenylyl-sulfate (PAPS) to sulfite. The protein has been cloned, overexpressed and purified from E. coli. Crystallization experiments resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C2221 with cell dimensions a = 81.9, h = 97.4, c = 109.5A, and contain one molecule per asymmetric unit. At cryogenic (100 K) temperatures the crystals diffract to a resolution limit of 2.7 A using a rotating anode and to 2.0 A at a synchrotron source.
Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3'-phosphotransferase) has bee... more Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3'-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B ("blue" or "red") and chromatofocusing on polybuffer PBE 94tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps. The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it disintegrated into assumingly identical smaller subunits (E. coli rom Mr 90-85,000 to 45-40,000 and S. cerevisiae from 52-49,500 to 28-29,500). Both forms, dimer and monomer were found catalytically active. Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.
Sulfite reductase (SIR) represents a key enzyme in sulfate assimilation in higher plants. The gen... more Sulfite reductase (SIR) represents a key enzyme in sulfate assimilation in higher plants. The genomic DNA sequence of the sir gene from Arabidopsis thaliana including regulatory and structural regions was isolated and characterized. The sequence of a 6 kb fragment encoding SIR revealed a coding region of 2891 basepairs (bp) that consists of eight exons separated by seven introns between 83 and 139 bp in length. The transcription start point was determined 272 bp upstream of the translation start site. Southern analysis indicates a single locus for the sir gene that gives rise to a 2.4 (kb) mRNA in leaves and in roots. The promoter region was verified by functional expression of the gusA reporter gene in transgenic A. thaliana plants and was shown to provide correct expression in root and leaf.
The first three enzymatic steps of assimilatory sulfate reduction in chloroplasts of higher plant... more The first three enzymatic steps of assimilatory sulfate reduction in chloroplasts of higher plants have been investigated with emphasis on the influence of adenosine-mono-and -diphosphate upon the formation of APS, PAPS and bound sulfite. The data show that the activation process is governed by the energy charge of the chloroplast. The regulatory step is localized at the ATP-sulfurylase reaction. It was found that this enzyme is inhibited by low concentrations of AMP and ADP, with apparent KiAMP = L8mм and KiADP=0.5 mм for the chloroplast preparations. The isolated purified ATP-sulfurylase is inhibited by the nucleotides accordingly, with KiAMP=0.2mм and KiADP=0.4mм. The results are interpreted as a regulatory mechanism for the complete process of assimilatory sulfate reduction in the chloro plast.
Plants assimilate inorganic sulphate for the bio synthesis of sulphur containing compounds. The e... more Plants assimilate inorganic sulphate for the bio synthesis of sulphur containing compounds. The enzymes required for reduction are found pre dominantly in the plastids which provide the necessary energy and the reductants. Sulphate re duction like nitrate or carbon dioxide assimilation is endergonic; for comparison, N 0 3 reduction to ammonia requires +347 kJ m ol-1 and C 0 2 re duction +478 kJ m ol-1 (Thauer et al., 1977). About twice as much energy is necessary to assimilate sulphate under aerobic conditions: SO I" + [8 H] + 2 H +-> H2S + 4 H 2 0. AG 0 = +733 kJ m ol-1.
Biosynthesis of the plant sulpholipid (diacylsulphoquinovosyl glycerol) has been demonstrated in ... more Biosynthesis of the plant sulpholipid (diacylsulphoquinovosyl glycerol) has been demonstrated in a cell-free assay system from the green alga Chlamydomonas reinhardii CW 15. The sulpholipid was labelled with [35S]sulphate when ATP, Mg2+ and a reductant were provided. Unlabelied cysteic acid failed to suppress the labelling of the sulphonate group in the lipid. Labelling with [35S]PAPS or [35S]sulphite has been studied in a fractionated cell extract. Sulphite as a precursor only required a particulate fraction of the cell while with PAPS the assay had to be complemented with a soluble protein and a thiol compound. [35S]PAPS was incorporated into the diacylsulphoquinovosyl glycerol with a higher specificity than sulphite.
Photoheterotrophic and heterotrophic cell suspension cultures of Nicotiana tabacum have been exam... more Photoheterotrophic and heterotrophic cell suspension cultures of Nicotiana tabacum have been examined for changes in specific activity of ATP-sulfurylase (EC 2.1.1 A) and OAS-sulfhydrylase (EC 4.2.99.8) during growth on different nitrogen sources. During exponential growth the specific activity of ATP-sulfurylase and OAS-sulfhydrylase remained constant and was on the same level in cells with high and with low rates of sulfate assimilation. The specific activity of both enzymes rapidly increased in green photoheterotrophic cells as well as in chloroplast-free heterotrophic cells after the sulfur from the medium had been used up. This increase was reversed when the cells were transfered back to a sulfate supplemented nutrient solution. The changes in enzyme activity due to sulfur depletion seem to indicate a regulatory mechanism for these enzymes.
PAPS-reductase from Escherichia coli was em ployed to detect thioredoxins from pro- and eukaryoti... more PAPS-reductase from Escherichia coli was em ployed to detect thioredoxins from pro- and eukaryotic organisms. A simple method for the isolation of this enzyme and properties of the enzymatic assay were described. A comparison betw een thioredoxins detected by the PAPS-reductase and the Fructose-bisphosphatase or NADP malate dehydrogenase was used to assess the validity of the test. The high cross-reactivity of the bacterial enzyme was useful in the purification of heterologous thioredoxins from spinach, Synechococcus, and Saccharomyces cerevisiae.
... Jens Dirk Schwenn, Brigitte Depka and Hagen Heinrich Hennies ... sulfate reduction was found ... more ... Jens Dirk Schwenn, Brigitte Depka and Hagen Heinrich Hennies ... sulfate reduction was found to be virtually uneffected by osmotic shock and the following dilution, which for comparison, completely eliminates photo-synthetic CC>2-fixation according to Stokes and Walker (27). ...
Specific long- and short-range electrostatic interactions and not redox potentials determine the ... more Specific long- and short-range electrostatic interactions and not redox potentials determine the substrate specificity of Trx family proteins.
Thioredoxin in the bacterial or mammalian cell is reduced by the NADPH: thioredoxin oxidoreductas... more Thioredoxin in the bacterial or mammalian cell is reduced by the NADPH: thioredoxin oxidoreductase (EC 1.6.4.5) [1,2]. The enzyme is a flavoprotein which carries FAD as the prosthetic group [3]. With the reduction of thioredoxin it supplies the hydrogen donor required for the formation of deoxynucleotides. In the photosynthetically active plant cell thioredoxins contribute to the regulation of enzyme activity [4]. In this respect, it may be regarded as a “non essential” activator. So far, three of these regulatory proteins have been identified as thioredoxin c, f and m. Excluding the cytoplasmic thioredoxin c which function still is unclear it is generally accepted that the f — and m-type of chloroplastic thioredoxin is reduced in a light-driven reaction via reduced ferredoxin [5] through the action of a ferredoxin: thioredoxin oxidoreductase [6]. This protein, in contrast to the NADP: thioredoxin oxidoreductase (EC 1.6.4.5) was found devoid of a chromophore. Evidence for a separate enzyme present in the leaves of higher plants has recently been obtained by Soulie et al [7]. They assumed that the NADPH dependent reduction of thioredoxin f very likely is catalyzed by the plant ferredoxin: NADP oxidoreductase (EC 1.18.1.2).
Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocysti... more Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803. One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over-expressed in E. coli and purified. The purified protein was found to be mainly dimeric and did not contain any cofactor. Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one [2Fe2S] cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and Mössbauer spectroscopy. Mössbauer spectroscopy further indicated that the FeS cluster was bound by four cysteine residues. Site-directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding. It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers. The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half-time of 10 min. Surprisingly, incubation of cluster-containing IscA with apo adenosine 5'-phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster. This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units.
Biosynthesis of PAPS by algal or higher plant material has occasionally been reported (references... more Biosynthesis of PAPS by algal or higher plant material has occasionally been reported (referencesa in Table 1) and, so far, three different types of enzymatic assays for the APS-kinase have been introduced (referencesb in Table 1). The presence of APS-kinase (EC 2.7.1.25, ATP: adenylylsulfate-3′-phosphotransferase) in plants seems to be ascertained. But, as compared to other enzymes involved in the assimilatory reduction of sulfate, detailed investigations of the APS-kinase are not available.
Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin-depen... more Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin-dependent reduction of 3'-phosphoadenylylsulphate (PAPS) to sulphite. The enzyme became detectable when heterologous thioredoxin from Escherichia coli was used substituting for the hitherto unidentified plant thioredoxin. The enzyme's cross-reactivity with heterologous thioredo xin enabled the partial purification and brief characterization. The molecular weight of the en zyme as estimated by HPLC size exclusion and gel filtration was 68-72 k. The protein reduced PAPS only when thioredoxin was present as cosubstrate. The function of this enzyme in the assimilation of inorganic sulphate by higher plants is discussed in comparison to the function of the respective enzymes from Escherichia coli and Saccharom yces cerevisiae.
Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 m... more Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared "repressed" by the S-amino acids but the GSH-dependent enzyme was "derepressed." In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells.
The structural genes of enterobacteria encoding for the enzymes involved in the assimilatory redu... more The structural genes of enterobacteria encoding for the enzymes involved in the assimilatory reduction of sulphate (“cys genes”) were used in order to identify homologous genes from phototrophic cyanobacteria and higher plants. By Southern hybridisation of genomic DNA from the higher organisms with the cys DNA-probes derived from Escherichia coli, discrete restriction fragments were found in higher plants and in cyanobacteria indicating the occurrence of related DNA. Two of the cyanobacterial genes were cloned and identified by DNA and amino acid sequence comparison as the structural genes encoding the PAPS-reductase (EC 1.8.4.-) and the ferredoxin: sulphite-reductase (EC 1.8.7.1). The nucleic acid of both genes showed stretches of highly conserved bases in regions of the sequences which are regarded as the functionally important domains of the gene products.
Tke common adenine nucleotides (adenosine mono-, di-, triphosphate) have been separated by paired... more Tke common adenine nucleotides (adenosine mono-, di-, triphosphate) have been separated by paired-ion chromatography from tkeir sulpkated derivatives (adenosine S-sulpkatopkospkate, adenosine 3'-phosphate S-sulphatophosphate). With tetrabutylammonium hydroxide as the ion-pair forming reagent, nicotinamideadenine dinucleotide phosphate, Gavin-adenine dinucleotide and adenosine 3'S'-bisphospkate were also separated from these nucleotides by rapid isocratic elution. The method is highly reliable as shown by the capacity factors (I?), and its compatibility with the requirements of a continuous-ff ow radio detection for 3*S-lakelled nucleotides is demonstrated. It has keen apptied to an investigation of tke kinetics of a PAPS sulpkotransferase reaction involved in kigker plant assimilative sulpkate reduction.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1994
A cDNA clone with an open reading flame of 831 nucleotides was isolated from a AZaplI-library of ... more A cDNA clone with an open reading flame of 831 nucleotides was isolated from a AZaplI-library of Arabidopsis thaliana. The nucleotide sequence of the cDNA is homologous to the APS-kinase genes from enterobacteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53.2%), Rh&obium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14: 57.1%). The polypeptide deduced from the plant APS-kinase cDNA is comprised of 276 amino acid residues with a molecular weight of 29 790. It contains an N-terminal extension of 77 amino acids. This extension includes a putative transit peptide of 37 residues separated from the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26 050 is predicted for the processed protein. The relatedness between bacterial, fungal and plant APS-kinase polypeptides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. meliloti), and 50.3% (Azospirillum brasilense). The plant polypeptide contains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxxGK. Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif described for PAPS dependent enzymes. The identity of the gene was confirmed by analyzing the function of the gene product. The putative transit peptide was deleted by PCR and the truncated gene was expressed in a pTacl vector system. A polypetide of MW 25761 could be induced by IPTG. The gene product was enzymatically active as APS-kinase. It produced PAPS from APS and ATP-the absence of ATP but supplemented with thiols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransferase is not a separate enzyme but identical with APS-kinase.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1973
1. It has been proposed that Mg 2+, inorganic pyrophosphate and a protein fraction which exhibits... more 1. It has been proposed that Mg 2+, inorganic pyrophosphate and a protein fraction which exhibits fructose-l,6-dlphosphatase activity may interact to regulate photosynthesis by isolated chloroplasts. 2. Evidence is presented which confirms the interaction and regulation but shows that these effects are indirectly attributable to pyrophosphatase activity rather than fructose-1,6-dlphosphatase 3. When provided with Mg 2+ and PP, the pyrophosphatase simply alters the proportions of orthophosphate and PP, m the reaction mixture. As the P, concentration is increased, it first stimulates and then mhxblts, the degree of inhiblt~on being enhanced by additional Mg 2 +. PPI ameliorates the inhibition, possibly by chelation of Mg 2 +. 4 It is concluded that the proposed regulation is ultimately governed by the P, concentration and the known relationship between P, uptake and those phosphate export across the chloroplast envelope.
Acta Crystallographica Section D Biological Crystallography, 1998
PAPS reductase from E. coli is involved in sulfur metabolism and catalyses the reduction of phosp... more PAPS reductase from E. coli is involved in sulfur metabolism and catalyses the reduction of phospho-adenylyl-sulfate (PAPS) to sulfite. The protein has been cloned, overexpressed and purified from E. coli. Crystallization experiments resulted in crystals suitable for X-ray diffraction. The crystals belong to the orthorhombic space group C2221 with cell dimensions a = 81.9, h = 97.4, c = 109.5A, and contain one molecule per asymmetric unit. At cryogenic (100 K) temperatures the crystals diffract to a resolution limit of 2.7 A using a rotating anode and to 2.0 A at a synchrotron source.
Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3'-phosphotransferase) has bee... more Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3'-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B ("blue" or "red") and chromatofocusing on polybuffer PBE 94tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps. The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it disintegrated into assumingly identical smaller subunits (E. coli rom Mr 90-85,000 to 45-40,000 and S. cerevisiae from 52-49,500 to 28-29,500). Both forms, dimer and monomer were found catalytically active. Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.
Sulfite reductase (SIR) represents a key enzyme in sulfate assimilation in higher plants. The gen... more Sulfite reductase (SIR) represents a key enzyme in sulfate assimilation in higher plants. The genomic DNA sequence of the sir gene from Arabidopsis thaliana including regulatory and structural regions was isolated and characterized. The sequence of a 6 kb fragment encoding SIR revealed a coding region of 2891 basepairs (bp) that consists of eight exons separated by seven introns between 83 and 139 bp in length. The transcription start point was determined 272 bp upstream of the translation start site. Southern analysis indicates a single locus for the sir gene that gives rise to a 2.4 (kb) mRNA in leaves and in roots. The promoter region was verified by functional expression of the gusA reporter gene in transgenic A. thaliana plants and was shown to provide correct expression in root and leaf.
The first three enzymatic steps of assimilatory sulfate reduction in chloroplasts of higher plant... more The first three enzymatic steps of assimilatory sulfate reduction in chloroplasts of higher plants have been investigated with emphasis on the influence of adenosine-mono-and -diphosphate upon the formation of APS, PAPS and bound sulfite. The data show that the activation process is governed by the energy charge of the chloroplast. The regulatory step is localized at the ATP-sulfurylase reaction. It was found that this enzyme is inhibited by low concentrations of AMP and ADP, with apparent KiAMP = L8mм and KiADP=0.5 mм for the chloroplast preparations. The isolated purified ATP-sulfurylase is inhibited by the nucleotides accordingly, with KiAMP=0.2mм and KiADP=0.4mм. The results are interpreted as a regulatory mechanism for the complete process of assimilatory sulfate reduction in the chloro plast.
Plants assimilate inorganic sulphate for the bio synthesis of sulphur containing compounds. The e... more Plants assimilate inorganic sulphate for the bio synthesis of sulphur containing compounds. The enzymes required for reduction are found pre dominantly in the plastids which provide the necessary energy and the reductants. Sulphate re duction like nitrate or carbon dioxide assimilation is endergonic; for comparison, N 0 3 reduction to ammonia requires +347 kJ m ol-1 and C 0 2 re duction +478 kJ m ol-1 (Thauer et al., 1977). About twice as much energy is necessary to assimilate sulphate under aerobic conditions: SO I" + [8 H] + 2 H +-> H2S + 4 H 2 0. AG 0 = +733 kJ m ol-1.
Biosynthesis of the plant sulpholipid (diacylsulphoquinovosyl glycerol) has been demonstrated in ... more Biosynthesis of the plant sulpholipid (diacylsulphoquinovosyl glycerol) has been demonstrated in a cell-free assay system from the green alga Chlamydomonas reinhardii CW 15. The sulpholipid was labelled with [35S]sulphate when ATP, Mg2+ and a reductant were provided. Unlabelied cysteic acid failed to suppress the labelling of the sulphonate group in the lipid. Labelling with [35S]PAPS or [35S]sulphite has been studied in a fractionated cell extract. Sulphite as a precursor only required a particulate fraction of the cell while with PAPS the assay had to be complemented with a soluble protein and a thiol compound. [35S]PAPS was incorporated into the diacylsulphoquinovosyl glycerol with a higher specificity than sulphite.
Photoheterotrophic and heterotrophic cell suspension cultures of Nicotiana tabacum have been exam... more Photoheterotrophic and heterotrophic cell suspension cultures of Nicotiana tabacum have been examined for changes in specific activity of ATP-sulfurylase (EC 2.1.1 A) and OAS-sulfhydrylase (EC 4.2.99.8) during growth on different nitrogen sources. During exponential growth the specific activity of ATP-sulfurylase and OAS-sulfhydrylase remained constant and was on the same level in cells with high and with low rates of sulfate assimilation. The specific activity of both enzymes rapidly increased in green photoheterotrophic cells as well as in chloroplast-free heterotrophic cells after the sulfur from the medium had been used up. This increase was reversed when the cells were transfered back to a sulfate supplemented nutrient solution. The changes in enzyme activity due to sulfur depletion seem to indicate a regulatory mechanism for these enzymes.
PAPS-reductase from Escherichia coli was em ployed to detect thioredoxins from pro- and eukaryoti... more PAPS-reductase from Escherichia coli was em ployed to detect thioredoxins from pro- and eukaryotic organisms. A simple method for the isolation of this enzyme and properties of the enzymatic assay were described. A comparison betw een thioredoxins detected by the PAPS-reductase and the Fructose-bisphosphatase or NADP malate dehydrogenase was used to assess the validity of the test. The high cross-reactivity of the bacterial enzyme was useful in the purification of heterologous thioredoxins from spinach, Synechococcus, and Saccharomyces cerevisiae.
... Jens Dirk Schwenn, Brigitte Depka and Hagen Heinrich Hennies ... sulfate reduction was found ... more ... Jens Dirk Schwenn, Brigitte Depka and Hagen Heinrich Hennies ... sulfate reduction was found to be virtually uneffected by osmotic shock and the following dilution, which for comparison, completely eliminates photo-synthetic CC>2-fixation according to Stokes and Walker (27). ...
Specific long- and short-range electrostatic interactions and not redox potentials determine the ... more Specific long- and short-range electrostatic interactions and not redox potentials determine the substrate specificity of Trx family proteins.
Thioredoxin in the bacterial or mammalian cell is reduced by the NADPH: thioredoxin oxidoreductas... more Thioredoxin in the bacterial or mammalian cell is reduced by the NADPH: thioredoxin oxidoreductase (EC 1.6.4.5) [1,2]. The enzyme is a flavoprotein which carries FAD as the prosthetic group [3]. With the reduction of thioredoxin it supplies the hydrogen donor required for the formation of deoxynucleotides. In the photosynthetically active plant cell thioredoxins contribute to the regulation of enzyme activity [4]. In this respect, it may be regarded as a “non essential” activator. So far, three of these regulatory proteins have been identified as thioredoxin c, f and m. Excluding the cytoplasmic thioredoxin c which function still is unclear it is generally accepted that the f — and m-type of chloroplastic thioredoxin is reduced in a light-driven reaction via reduced ferredoxin [5] through the action of a ferredoxin: thioredoxin oxidoreductase [6]. This protein, in contrast to the NADP: thioredoxin oxidoreductase (EC 1.6.4.5) was found devoid of a chromophore. Evidence for a separate enzyme present in the leaves of higher plants has recently been obtained by Soulie et al [7]. They assumed that the NADPH dependent reduction of thioredoxin f very likely is catalyzed by the plant ferredoxin: NADP oxidoreductase (EC 1.18.1.2).
Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocysti... more Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803. One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over-expressed in E. coli and purified. The purified protein was found to be mainly dimeric and did not contain any cofactor. Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one [2Fe2S] cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and Mössbauer spectroscopy. Mössbauer spectroscopy further indicated that the FeS cluster was bound by four cysteine residues. Site-directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding. It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers. The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half-time of 10 min. Surprisingly, incubation of cluster-containing IscA with apo adenosine 5'-phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster. This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units.
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