Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

In vivo molybdate inhibition of sulfate transfer to porphyridium capsular polysaccharide

Active transport of exogenous sulfate into log phase cells of Porphyridium aerueineum followed Michaelis-Menten kinetics, and the apparent Km for sulfate transport is approximately 2.5 × 10⁻⁶m. Molybdate, also a group VI anion, is a competitive inhibitor of sulfate transport, and the inhibition is freely reversible. Once in the cell, molybdate depresses the rate of sulfate pool utilization by blocking sulfate transfer to polysaccharides destined for secretion to the cell surface. Specifically, molybdate inhibits the formation of adenosine 5′-phosphosulfate and in turn the formation of adenosine 3′-phosphate 5′-phosphosulfate, the activated donor for sulfate transfer reactions. Combined with the previous identification of adenosine 3′-phosphate 5′-phosphosulfate, this is taken as evidence that the adenosine 5′-phosphosulfate/adenosine 3′-phosphate 5′-phosphosulfate enzymatic sequence for sulfate activation and sulfate donor reactions is operating in Porphyridium. Thiosulfate is utilized as effectively as sulfate as both a sulfur source for growth and polysaccharide synthesis.     

Regulation of Sulfate Assimilation in Plants: 7. Cysteine Inactivation of Adenosine 5'-Phosphosulfate Sulfotransferase in Lemna minor L

When 0.5 mm cysteine is added to cultures of Lemna minor L. growing with sulfate as the sole sulfur source, there is a rapid 80% loss of extractable adenosine 5′-phosphosulfate sulfotransferase. This loss is accompanied by an inhibition of sulfate uptake; however, lack of sulfate is not responsible for the decreasing adenosine 5′-phosphosulfate sulfotransferase activity.     

Regulation of Assimilatory Sulfate Reduction by Cadmium in Zea mays L

Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from ³⁵SO₄²⁻ into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5′-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5′-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves.     

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 ³⁵SO₄²⁻, 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 ³⁵SO₄²⁻, 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.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.     

Regulation of Sulfate Assimilation in Plants : XIII. Assimilatory Sulfate Reduction during Ontogenesis of Primary Leaves of Phaseolus vulgaris L

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of ³⁵SO₄²⁻ 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 ³⁵SO₄²⁻ 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 CO₂ and nitrate assimilation.     

Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L

The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5′-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5′-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of ³⁵SO₄²⁻ into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5′-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from ³⁵SO₄²⁻ into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation.     

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

Biochemical 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 K_m for adenosine 5′-phosphosulfate (APS) was 29 ± 5.5μM, its apparent M_r was 115,000. 5′-AMP inhibited the enzyme competitively with a K_i 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 S^2- up to 5mM cysteine and up to 200 mM SO₃^2- 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 adenosine 5'-phosphosulfate sulfotransferase by sulfur dioxide in primary leaves of beans (Phaseolus vulgaris)

SO₂ inhibited the light-induced increase of extractable adenosine 5′-phosphosulfate sulfotransferase in greening primary leaves of bean seedlings (Phaseolus vulgaris L. cv. Saxa (Radio) Stamm Vatter). In green primary leaves containing appreciable extractable adenosine 5′-phosphosulfate sulfotransferase activity, SO₂ treatment for 20 h decreased the activity of the enzyme to between 10 and 20% of the initial level. After removal of SO₂ from the air, the extractable adenosine 5′-phosphosulfate sulfotransferase activity increased after a lag, both in green and greening primary leaves, and was back to the control level after about 48 h. The sulfate concentration was increased about fourfold during SO₂ treatment. An increase in sulfate sulfur accompanied by a decrease in adenosine 5′-phosphosulfate sulfotransferase was also observed when bean seedlings, after excision of the roots, were transferred to nutrient solutions containing high sulfate concentrations, suggesting that sulfate is involved in the regulation of the enzyme.     

Analysis of the regulation of adenosine 5′-phosphosulfate sulfotransferase activity inLemna minor L. using15N-density labeling

The role of de novo synthesis in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S inLemna minor L. was investigate using density labeling with¹⁵N applied as¹⁵NO ₃ ^– in the culture medium. While adenosine 5-phosphosulfate sulfotransferase activity was rapidly reduced by H₂S and rapidly recovered upon removal of H₂S, O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) did not show changes in extractable activity in response to H₂S and could therefore be used as an internal marker enzyme for density labeling. The incorporation of¹⁵N into adenosine 5-phosphosulfate sulfotransferase was strongly reduced upon transfer of plants into a H₂S-containing atmosphere. Half-maximal labeling was reached only after 70–80 h compared to 40–50 h in the control. After removal of H₂S, adenosine 5-phosphosulfate sulfotransferase activity increased to the initial level within 20 h, and the enzyme reached halfmaximal labeling after only 15 h. The time course of the density increase of O-acetyl-L-serine sulfhydrylase was not affected very significantly by H₂S. These results provide evidence that de novo synthesis of enzyme protein is involved in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumine - DTE dithioerythritol - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - POPOP 1,4-bis-(5-phenyl-2-oxazolyl)-benzene - PPO 2,5-diphenyloxazole This is no. 9 in the series Regulation of Sulfate Assimilation in Plants     

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 ³⁵SO 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 ³⁵SO 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.Abbreviation APSSTase adenosine 5-phosphosulfate sulfotransferase     

Selenium Metabolism in Neptunia amplexicaulis

ATP sulfurylase (EC 2.7.7.4), cysteinyl-tRNA synthetase (EC 6.1.1.16), and methionyl-tRNA synthetase (EC 6.1.1.10) from Neptunia amplexicaulis have been purified approximately 162-, 140- and 185-fold, respectively. Purified ATP sulfurylase in the presence of purified inorganic pyrophosphatase catalyzed the incorporation of sulfate into adenosine 5′-phosphosulfate; evidence of an analogous reaction with selenate is presented. Crude extracts catalyzed both the sulfate- and the adenosine 5′-phosphosulfate-dependent NADH oxidation in the adenosine 5′-phosphosulfate kinase assay of Burnell and Whatley (1977 Biochim Biophys Acta 481: 266-278), but an analogous reaction with selenate could not be detected. Both purified cysteinyl-tRNA synthetase and methionyl-tRNA synthetase used selenium-containing analogs as substrates in both the ATP-pyrophosphate exchange and the aminoacylation assays.     

Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH₄)₂SO₄ precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent K_m for the last-mentioned compound is 0.82mm. The apparent K_m for ammonia (assuming NH₃ to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Adenosine 5'-triphosphate-sulfurylase in corn roots and its partial purification

ATP-sulfurylase (ATP-sulfate adenyltransferase, EC 2.7.7.4) was found in nonparticulate fractions of both roots and leaves of Zea mays L. seedlings using two detection methods. Addition of exogenous pyrophosphatase was essential for maximum rates of conversion of ³⁵SO₄²⁻ to labeled adenosine phosphosulfate in unpurified root extracts, but not in unpurified leaf extracts. In the presence of exogenous pyrophosphatase, the enzyme from roots exhibited specific activities as high as those obtained with the leaf enzyme. The root enzyme was purified 33-fold by centrifugation and column chromatography procedures. Its molecular weight obtained by Sephadex gel filtration was about 42,000. Its Km for pyrophosphate was 7 μm, while for adenosine phosphosulfate, the Km was 1.35 μm. None of the enzyme fractions studied converted adenosine phosphosulfate into detectable amounts of 3′-phosphoadenosine-5′-phosphosulfate. ATP-sulfurylase was also found in roots of corn seedlings grown aseptically. The data suggest that at least the first reaction in sulfate reduction might proceed as effectively in roots as in shoots.     

Redox-linked Gating of Nucleotide Binding by the N-terminal Domain of Adenosine 5′-Phosphosulfate Kinase

Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine 5′-phosphosulfate (APS) to 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Crystallographic studies of APSK from Arabidopsis thaliana revealed the presence of a regulatory intersubunit disulfide bond (Cys⁸⁶–Cys¹¹⁹). The reduced enzyme displayed improved catalytic efficiency and decreased effectiveness of substrate inhibition by APS compared with the oxidized form. Here we examine the effect of disulfide formation and the role of the N-terminal domain on nucleotide binding using isothermal titration calorimetry (ITC) and steady-state kinetics. Formation of the disulfide bond in A. thaliana APSK (AtAPSK) inverts the binding affinities at the ATP/ADP and APS/PAPS sites from those observed in the reduced enzyme, consistent with initial binding of APS as inhibitory, and suggests a role for the N-terminal domain in guiding nucleotide binding order. To test this, an N-terminal truncation variant (AtAPSKΔ96) was generated. The resulting protein was completely insensitive to substrate inhibition by APS. ITC analysis of AtAPSKΔ96 showed decreased affinity for APS binding, although the N-terminal domain does not directly interact with this ligand. Moreover, AtAPSKΔ96 displayed reduced affinity for ADP, which corresponds to a loss of substrate inhibition by formation of an E·ADP·APS dead end complex. Examination of the AtAPSK crystal structure suggested Arg⁹³ as important for positioning of the N-terminal domain. ITC and kinetic analysis of the R93A mutant also showed a complete loss of substrate inhibition and altered nucleotide binding affinities, which mimics the effect of the N-terminal deletion. These results show how thiol-linked changes in AtAPSK alter the energetics of binding equilibria to control its activity.     

Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5′-Phosphosulfate Kinase from Cyanobacteria to Plants

In plants, adenosine 5′-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants.     

Properties and regulation of adenosine 5′-phosphosulfate sulfotransferase from suspension cultures ofNicotiana sylvestris

The properties and the regulation of adenosine 5-phosphosulfate sulfotransferase extracted from cell suspension cultures ofNicotiana sylvestris was investigated. Optimal adenosine 5-phosphosulfate sulfotransferase activity was obtained from the cells by extraction with 0.1 M tris-HCl, pH8.0, containing 2 M MgSO₄ and 10 mM dithioerythritol. The K _m for adenosine 5-phosphosulfate in the sulfotransferase reaction was about 11 M. Adenosine 5-phosphosulfate in concentrations above 50 M were inhibitory. The extratable adenosine 5-phosphosulfate sulfotransferase activity decreased during cultivation with sulfate as the sole sulfur source, but after about 3 days it reached a constant level (50 to 100 nmol activated sulfate transferred h^-1 mg^-1 protein) which was maintained for at least 24 h. Addition of 0.5 mM cysteine to the culture medium decreased the extractable adenosine 5-phosphosulfate sulfotransferase activity and blocked growth completely. With 0.1 mM cysteine an enzyme level of about 10% of the initial value was reached within 6 to 12 h without significant inhibition of growth. The added cysteine was absorbed rapidly and after 24 h cysteine could no longer be detected in the medium. Before the cysteine was completely depleted, the activity of adenosine 5-phosphosulfate sulfotransferase started to increase, reaching ultimately a level which was comparable to the initial value.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - DTE dithioerythritol - PAPS adenosine 3-phosphate 5-phosphosulfate - 2,4-D 2,4-di-chlorophenoxyacetic acid - BAP benzyladenine This paper is no. 10 in the series Regulation of Sulfate Assimilation in Plants.