Jens D. Schwenn

ISOLATION AND CHARACTERIZATION OF A GENE FOR ASSIMILATORY SULFITE REDUCTASE FROM ARABIDOPSIS THALIANA

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.

Characterization and Reconstitution of a 4Fe-4S Adenylyl Sulfate/ Phosphoadenylyl Sulfate Reductase from Bacillus subtilis*

CysH1 from Bacillus subtilis encodes a 3′-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa. Recombinant B. subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3′-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants. The enzyme has a higher affinity for PAPS (KmPAPS 6.4 μm Trx-saturating, 10.7 μm Grx-saturating) than for APS (Km APS 28.7 μm Trx-saturating, 105 μm Grx-saturating) at a Vmax ranging from 280 to 780 nmol sulfite mg-1 min-1. The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (Kcat/Km 2.7 × 104-3.6 × 104 liter mol-1 s-1. B. subtilis SNR contains one 4Fe-4S cluster per polypeptide chain. SNR activity and color were lost rapidly upon exposure to air or upon dilution. Mössbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster. Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS. Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS. The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO3 formation under aerobiosis.

Yeast PAPS reductase: properties and requirements of the purified enzyme

The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3′-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (MR 80–85 k) is represented by a dimer which reduces 3′-phosphoadenylyl sulphate to adenosine-3′,5′-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for “bound-sulphite(s)” as intermediate. Vmax of the enriched enzyme was 4–7 nmol sulphite · min-1 · mg-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite · min-1 · mg-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The Km of homologous thioredoxin was 0.6 · 10-6 M, for the heterologous cosubstrate it increased to 1.4 · 10-6 M. The affinity for PAPS remained practically unaffected (Km PAPS: 19 · 10-6 M in the homologous, and 21 · 10-6 M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.

Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide α hydrolases

BACKGROUND Assimilatory sulphate reduction supplies prototrophic organisms with reduced sulphur for the biosynthesis of all sulphur-containing metabolites. This process is driven by a sequence of enzymatic steps involving phosphoadenylyl sulphate (PAPS) reductase. Thioredoxin is used as the electron donor for the reduction of PAPS to phospho-adenosine-phosphate (PAP) and sulphite. Unlike most electron-transfer reactions, there are no cofactors or prosthetic groups involved in this reduction and PAPS reductase is one of the rare examples of an enzyme that is able to store two electrons. Determination of the structure of PAPS reductase is the first step towards elucidating the biochemical details of the reduction of PAPS to sulphite. RESULTS We have determined the crystal structure of PAPS reductase at 2.0 A resolution in the open, reduced form, in which a flexible loop covers the active site. The protein is active as a dimer, each monomer consisting of a central six-stranded beta sheet with alpha helices packing against each side. A highly modified version of the P loop, the fingerprint peptide of mononucleotide-binding proteins, is present in the active site of the protein, which appears to be a positively charged cleft containing a number of conserved arginine and lysine residues. Although PAPS reductase has no ATPase activity, it shows a striking similarity to the structure of the ATP pyrophosphatase (ATP PPase) domain of GMP synthetase, indicating that both enzyme families have evolved from a common ancestral nucleotide-binding fold. CONCLUSIONS The sequence conservation between ATP sulphurylases, a subfamily of ATP PPases, and PAPS reductase and the similarities in both their mechanisms and folds, suggest an evolutionary link between the ATP PPases and PAPS reductases. Together with the N type ATP PPases, PAPS reductases and ATP sulphurylases are proposed to form a new family of homologous enzymes with adenine nucleotide alpha-hydrolase activity. The open, reduced form of PAPS reductase is able to bind PAPS, whereas the closed oxidized form cannot. A movement between the two monomers of the dimer may allow this switch in conformation to occur.

A cDNA clone from Arabidopsis thaliana encoding plastidic gerredoxin: sulfite reductase☆

A cDNA with an open reading frame of 1929 bp (termed sir) was isolated from a lambda ZapII library of Arabidopsis thaliana leaf tissue. The polypeptide sequence deduced from the cDNA is homologous to the ferredoxin-dependent sulfite reductase (EC 1.8.7.1) from Synechococcus PCC7942 and distantly related to the hemoprotein subunit of Escherichia coli NADPH-dependent sulfite reductase (EC 1.8.1.2). A molecular mass of 71.98 kDa can be predicted for a ferredoxin sulfite reductase from A. thaliana. The polypeptide consists of 642 amino acids including a transit peptide of 66 residues (6.72 kDa) that is assumed to direct the protein into the plastid. For expression and enzymatic characterization of a putative A. thaliana ferredoxin sulfite reductase, the DNA of the transit peptide was deleted by a PCR method. The truncated cDNA clone was expressed as his-tag fusion protein. The modified gene product was enzymatically inactive but specific cross-reaction with polyclonal antibodies against ferredoxin sulfite reductase from Synechococcus is seen as confirmation of its identity as higher plant ferredoxin sulfite reductase.

Characterisation of the gene cysH and of its product phospho-adenylylsulphate reductase from Escherichia coli

SummaryThe nucleotide sequenceThese sequence data will appear in the EMBL Sequence Data Library under the accession number Y07525 of the gene cysH from Escherichia coli K12 was determined. The open reading frame was 735 nucleotides in length; it was flanked by a repetitive palindromic sequence centred 36 nucleotides upstream of cysH and a terminator-like structure located 20 nucleotides downstream. CysH encoded a colypeptide of Mr 27927 consisting of 244 amino acids. The gene product was isolated as a homodimer exhibiting phospo-adenylylsulphate reductase (PAPS reductase) activity. The active enzyme was devoid of electron transferring cofactors and contained only one cysteine per subunit. Reduction of the enzyme by dithiols resulted in a shift of the apparent molecular weight from 44000 to 62000 without formation of an enzyme-thioredoxin complex.

Structural and kinetic properties of adenylyl sulfate reductase from Catharanthus roseus cell cultures.

A cDNA encoding a plant-type APS reductase was isolated from an axenic cell suspension culture of Catharanthus roseus (Genbank/EMBL-databank accession number U63784). The open reading frame of 1392 bp (termed par) encoded for a protein (Mr=51394) consisting of a N-terminal transit peptide, a PAPS reductase-like core and a C-terminal extension with homology to the thioredoxin-like domain of protein disulfide isomerase. The APS reductase precursor was imported into pea chloroplasts in vitro and processed to give a mature protein of approximately 45 kDa. The homologous protein from pea chloroplast stroma was detected using anti:par polyclonal antibodies. To investigate the catalytical function of the different domains deleted par proteins were purified. ParDelta1 lacking the transit sequence liberated sulfite from APS (Km 2.5+/-0.23 microM) in vitro with glutathione (Km 3+/-0.64 mM) as reductant (Vmax 2.6+/-0.14 U mg-1, molecular activity 126 min-1). ParDelta2 lacking the transit sequence and C-terminal domain had to be reconstituted with exogenous thioredoxin as reductant (Km 15. 3+/-1.27 microM, Vmax 0.6+/-0.014 U mg-1). Glutaredoxin, GSH or DTT were ineffective substitutes. ParDelta1 (35.4%) and parDelta2 (21. 8%) both exhibited insulin reductase activity comparable to thioredoxin (100%). Protein disulfide isomerase activity was observed for parDelta1.

Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae

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 desintegrated into assumingly identical smaller subunits (E. coli rom Mr 90-85000 to 45-40000 and s. cerevisiae from 52-49500 to 28-29500). 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.

A cDNA for adenylyl sulphate (APS)-kinase from Arabidopsis thaliana

A cDNA clone with an open reading frame of 831 nucleotides was isolated from a lambda ZapII-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%), Rhizobium 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 pTac1 vector system. A polypeptide 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.

A new role for thioredoxin in assimilatory sulphate reduction: Activation of the adenylylsulphate kinase from the green alga Chlamydomonas reinhardii CW 15

The activity of isolated APS‐kinase (EC 2.7.1.25) from the green alga Chlamydomonas reinhardii CW15 was stimulated by catalytic amounts of spinach thioredoxin f. The stimulation amounted to a factor of 3 in the presence of excess reducing thiols. A regulatory function of thioredoxin f was deduced from two observations: (a) the enzyme supported a low basal rate of PAPS formation in the absence of its effector, and (b) the saturation of the enzyme activity by thioredoxin f was sigmoidal. No evidence has been obtained of thioredoxin f serving as in the reduction of PAPS to sulphite in this alga. The slight increase in sulphite formation as observed with enriched spinach thioredoxin m is assumed to be due to a residual APS‐kinase contaminating the partially purified sulpho‐nucleotide unspecific sulphotransferase.