Enzymes Of Assimilatory Sulfate Reduction Research Articles

Adenosine-5'-Phosphosulfate- and Sulfite Reductases Activities of Sulfate-Reducing Bacteria from Various Environments.

A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282–0.862 µmol/min×mg−1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg−1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg−1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction—sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.

Influence of Chilling Stress on the Intercellular Distribution of Assimilatory Sulfate Reduction and Thiols in Zea mays

Abstract: The effect of chilling on the intercellular distribution of mRNAs for enzymes of assimilatory sulfate reduction, the activity of adenosine 5′‐phosphosulfate reductase (APR), and the level of glutathione was analysed in leaves and roots of maize (Zea mays L). At 25 °C the mRNAs for APR, ATP sulfurylase, and sulfite reductase accumulated in bundle‐sheath only, whereas the mRNA for O‐acetylserine sulfhydrylase was also detected in mesophyll cells. Glutathione was predominantly detected in mesophyll cells; however, oxidized glutathione was equally distributed between the two cell types. Chilling at 12 °C induced oxidative stress which resulted in increased concentrations of oxidized glutathione in both cell types and a prominent increase of APR mRNA and activity in bundle‐sheath cells. After chilling, mRNAs for APR and sulfite reductase, as well as low APR activity, were detected in mesophyll cells. In roots, APR mRNA and activity were at higher levels in root tips than in the mature root and were greatly increased after chilling. These results demonstrate that chilling stress affected the levels and the intercellular distribution of mRNAs for enzymes of sulfate assimilation.

Sulfate assimilation in higher plants characterization of a stable intermediate in the adenosine 5'-phosphosulfate reductase reaction.

The enzyme catalysing the reduction of adenosine 5'-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 degrees C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2-3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5'-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 degrees C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2-3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2-3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

Adenosine 5′-Phosphosulfate Sulfotransferase and Adenosine 5′-Phosphosulfate Reductase Are Identical Enzymes

Adenosine 5'-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M(r) of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K(m) = 6.5 microM) and not adenosine 3'-phosphate 5'-phosphosulfate as sulfonyl donor. The V(max) of recombinant Lemna APS sulfotransferase (40 micromol min(-1) mg protein(-1)) was about 10 times higher than the previously published V(max) of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO(3)(2-). Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO(3)(2-) was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Effect of Chilling on Assimilatory Sulfate Reduction and Glutathione Synthesis in Maize

The contents of cysteine, γ-glutamylcysteine and glutathione, as well as the activity of enzymes of assimilatory sulfate reduction and glutathione synthesis in second leaves and root tips of maize seedlings ( Zea mays L. cv. LG 11) cultivated at low temperatures, were compared with controls grown at 25 °C. In vitro nitrate reductase (EC 1.6.6.1) activity was measured for comparison. Compared with the controls, adenosine 5'-phosphosulfate sulf otransf erase activity was 3.5- and 5.5-fold higher at 12 °C in second leaves and root tips, respectively. After 3 days of growth, the activity of y,-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in the second leaves was also significantly higher at 12 °C compared with 25 °C. Consistent with these enzyme activities, there was an increase in cysteine and glutathione contents both in second leaves and root tips after 3 days of growth at 12 °C. Nitrate reductase activity of second leaves and root tips was not affected by growth at 12 °C, indicating that the increase in adenosine 5'-phosphosulfate sulfotransferase activity and of the enzymes of glutathione synthesis was specific. Our results demonstrate that the increased activity of a key enzyme of assimilatory sulfate reduction and of the enzymes of glutathione synthesis contribute to the increased glutathione levels measured at 12 °C.

Adenosine 5‘‐Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Properties*

AbstractBiochemical and physiological properties of adenosine 5′‐phosphosulfate sulfotransferase, a key enzyme of assimilatory sulfate reduction, from spruce trees growing under field conditions were studied. The apparent Km for adenosine 5′‐phosphosulfate (APS) was 29 ± 5.5μM, its apparent Mr was 115,000. 5′‐AMP inhibited the enzyme competitively with a Ki of 1 mM, but also stabilized it. MgS04 at 800 mM increased adenosine 5′‐phosphosulfate sulfotransferase activity by a factor of 3, concentrations higher than lOOOmM were inhibitory. Treatment of isolated shoots with nutrient solution containing 1 or 2 mM sulfate, and 3 or 10 mM glutathione, respectively, induced a significant decrease in extractable adenosine 5′‐phosphosulfate sulfotransferase activity over 24h, whereas GSH as well as S2‐ up to 5mM cysteine and up to 200 mM SO32‐ had no effect on the in vitro activity of the enzyme. As with other enzymes involved in assimilatory sulfate reduction, namely ATP sulfurylase (EC 2.7.7.4), sulfite reductase (EC 1.8.7.1) and O‐acetyl‐L.‐serine sulfhydrylase (EC 4.2.99.8), adenosine 5′‐phosphosulfate sulfotransferase was still detected at appreciable activities in 2‐ and 3‐year‐old needles. Adenosine 5′‐phosphosulfate sulfotransferase activity was low in buds and increased during shoot development, parallel to the chlorophyll content. The enzyme activity was characterized by an annual cycle of seasonal changes with an increase during February and March.

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L.

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of (35)SO(4) (2-), and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using (35)SO(4) (2-), roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5'-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5'-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5'-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.

Localization of enzymes of assimilatory sulfate reduction in pea roots

The localization of enzymes of assimilatory sulfate reduction was examined in roots of 5-d-old pea (Pisum sativum L.) seedlings. During an 8-h period, roots of intact plants incorporated more label from (35)SO 4 (2-) in the nutrient solution into the amino-acid and protein fractions than shoots. Excised roots and roots of intact plants assimilated comparable amounts of radioactivity from (35)SO 4 (2-) into the amino-acid and protein fractions during a 1-h period, demonstrating that roots of pea seedlings at this stage of development were not completely dependent on the shoots for reduced sulfur compounds. Indeed, these roots contained activities of ATP-sulfurylase (EC 2.7.7.4), adenosine 5'-phosphosulfate sulfotransferase, sulfite reductase (EC 1.8.7.1) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) at levels of 50, 30, 120 and 100%, respectively, of that in shoots. Most of the extractable activity of adenosine 5'-phosphosulfate sulfotransferase was detected in the first centimeter of the root tip. Using sucrose density gradients for organelle separation from this part of the root showed that almost 40% of the activity of ATP-sulfurylase, adenosine 5'-phosphosulfate sulfotransferase and sulfite reductase banded with the marker enzyme for proplastids, whereas only approximately 7% of O-acetyl-L-serine sulfhydrylase activity was detected in these fractions. Because their distributions on the gradients were very similar to that of nitrite reductase, a proplastid enzyme, it is concluded that ATP-sulfurylase, adenosine 5'-phosphosulfate sulfotransferase and sulfite reductase are also exclusively or almost exclusively localized in the proplastids of pea roots. O-Acetyl-L-serine sulfhydrylase is predominantly present in the cytoplasm.

Enzymes of assimilatory sulfate reduction in leaves of Pisum sativum: Activity changes during ontogeny and in vivo regulation by H2S and cyst(e)ine

Enzyme activities of assimilatory sulfate reduction were measured in leaves of Pisum sativum L., cv. Vatters Frühbusch, during their ontogenetic development, and during treatment with H2S and cyst(e)ine. Ribulose bisphosphate (RuBP) carboxylase (EC 4.1.1.39) and ferredoxin‐dependent nitrite reductase (Fd‐NiR, EC 1.7.7.1) were measured for comparison. In etiolated pea leaves, ATP‐sulfurylase (ATPase, EC 2.7.7.4), adenosine 5′‐phosphosulfate sulfotransferase (APSSTase), ferredoxin‐dependent sulfite reductase (Fd‐SiR, EC 1.8.7.1) and O‐acetyl‐L‐serine sulfhydrylase (OASSase, EC 4.2.99.8) activities were measured in appreciable rates, while neither RuBP carboxylase nor Fd‐NiR activities could be detected.During the first 2–7 days after transfer into the light all enzyme activities increased. After reaching maximal activities, ATPase, APSSTase, and Fd‐SiR activities decreased in all leaves to low or indetectable levels during the following 3–6 days. RuBP carboxylase, Fd‐NiR and OASSase, on the other hand, decreased slowly and were still at high levels of activity at the end of the experiment.Fumigation of pea plants with 1.5 μl l−1 H2S delayed the initial increase and the subsequent decrease of ATPase activity by 1–3 days. APSSTase activity decreased for 1–2 days, increased rapidly during the next 4–6 days and retained a high level of activity until the end of the experiment as did Fd‐SiR. One to two days after the beginning of fumigation the leaves started to accumulate high amounts of cyst(e)ine.When pea plants with excised roots were placed on a nutrient solution containing cyst(e)ine, APSSTase activity decreased more on 0.2 and 0.5 mM than on 1.0 mM. Fd‐SiR activity was only slightly decreased on 1.0 mM cyst(e)ine. Neither Fd‐NiR nor RuBP carboxylase activities were affected.

Regulation of sulfate assimilation by amino acids in Lemna minor L.

The effect of 2 mM arginine, asparagine and glutamine in the culture medium of Lemna minor L. on adenosine 5′-phosphosulfate sulfotransferase (APSSTase), a key enzyme of assimilatory sulfate reduction, was examined. These amino acids caused a 50–110% increase in extractable enzyme activity on a fresh weight basis. With asparagine and glutamine applied together at 2 mM this increase started 2 h after additionn and was completed after 6 h. Omission of these amino acids caused a decrease in APSSTase activity and by 24 h the original level was attained. ATP sulfurylase activity (EC 2.7.7.4) was not affected significantly by 24 h of cultivation of Lemna in the presence of amino acids and was consistently lower after 72 h than in controls which had nitrate as the sole nitrogen source. The extractable proteins were 10–30% higher in plants cultivated with amino acids for 72 h. 15N-density labelling experiments showed that the increase in APSSTase acitivity was predominantly or, perhaps, exclusively due to an increased synthesis, de novo. This regulation of APSSTase can be explained by assuming a mechanism aimed at producing sufficient amounts of sulfur amino acids during a period of increased protein synthesis.

Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L.

The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty-four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH(4) (+) added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH(4) (+), the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH(4) (+) from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO(3) (-) as a nitrogen source. Using [(35)S]SO(4) (2-) as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH(4) (+) was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH(4) (+) may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis.

Activity of Sulfate-assimilating Enzymes in Primary Leaves of Phaseolus vulgaris L. During Dark-induced Senescence

Summary The activity of three enzymes of assimilatory sulfate reduction, ATP sulfurylase, adenosine 5′-phosphosulfat sulfotransferase, and O-acetyl-L-serine sulfhydrylase, was measured in extracts from primary leaves of Phaseolus vulgaris L. seedlings during dark-induced senescence. When 7-day-old seedlings were transferred from continuous light to darkness there was an increase in total activity of ATP sulfurylase (E.C. 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase during the first 24 h. This was followed by a rapid decrease. After 6 days in the dark both enzyme activities were no longer detectable. Ribulosebisphosphate carboxylase activity, measured for comparison, increased during the first 3 days in the dark and remained constant during the following 3 days. When the identical experiment was performed with 10-day-old seedlings, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase activity started decreasing already during the first 24 h in the dark with a halflife of 40 and 20 h, respectively. The half-life of ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase activity in control seedlings kept in continuous light was 52 and 50 h, respectively. Ribulosebisphosphate carboxylase activity and chlorophyll content decreased at about the same rate in seedlings kept in the light or in the dark with half-lives of more than 100 h. In excised primary leaves from 10-day-old seedlings, the dark-induced decrease in activity of ATP sulfurylase, adenosine 5'-phosphosulfate sulfotransferase, and ribulosebisphosphate carboxylase and the content of chlorophyll was similar to that in leaves of intact plants . Total O-acetyl-L-serine sulfhydrylase activity only decreased by about 30% in leaves of 10-day-old seedlings transferred to the dark for 7 days. The O-acetyl-L-serine sulfhydrylase activity associated with the chloroplasts decreased more rapidly than the extrachloroplastic activity.

Regulation of Sulfate Assimilation in Plants

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of (35)SO(4) (2-) into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5'-phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of (35)SO(4) (2-) into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5'-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO(2) and nitrate assimilation.

Regulation of enzymes of assimilatory sulfate reduction in aerated cell suspension cultures of Nicotiana sylvestris

The regulation of ATP sulfurylase (EC 2.7.7.4), adenosine 5′-phosphosulfate sulfotransferase (APSSTase) and O-acetyl-L-serine sulfhydrylase (OASSase, EC 4.2.99.8) by H 2S and cysteine in aerated cell suspension cultures of Nicotiana sylvestris was investigated. Aeration with 6 μl · 1 −1 H 2S or addition of 0.1 mM cysteine to the culture medium did not affect significantly ATP sulfurylase and OASSase activity or the doubling time of the cells, but caused a rapid decrease in extractable APSSTase activity. Parallel to the decrease in APSSTase activity in H 2S treated cells there was an increase in the cysteine content of the cells. Both effects were readily reversible after omission of H 2S. The uptake and the reduction of radioactive sulfate were inhibited in cells containing low APSSTase activity.