Sulfurylase Activity Research Articles

Nitrate enhances cadmium accumulation through modulating sulfur metabolism in sweet sorghum

Sweet sorghum deploys tremendous potential for phytoremediation of cadmium (Cd)-polluted soils. Nitrate increases Cd accumulation in sweet sorghum, but the mechanism underlying this is still elusive. Sulfur-containing metabolites have been corroborated to play important roles in Cd tolerance in plants. Thus, whether sulfur metabolism contributed to nitrate-increased Cd accumulation in sweet sorghum was investigated in the present study. Two-way ANOVA analysis showed that most sulfur-containing metabolites concentrations and relevant enzymes activities were regulated by nitrate, Cd and interplay of nitrate and Cd. By using grey correlation analysis and Pearson correlation coefficient, Cd accumulation in shoots as affected by nitrate was also mainly ascribed to sulfur metabolism. ATP sulfurylase (ATPS) activities and non-protein thiol (NPT) concentrations in leaves were the two prominent factors that positively correlated with Cd accumulation in shoots. Excess nitrate elevated ATPS activities in leaves which contributed to increased NPT and phytochelatins (PCs) concentrations in leaves. Nitrate enhanced Cd accumulation in shoots of sweet sorghum under a low level of Cd treatment. Intriguingly, Cd accumulation in shoots of sweet sorghum was similar between a low level and a high level of Cd treatment. Principal Components Analysis (PCA) based on 34 parameters failed to separate the low Cd treatment from the high Cd treatment either, suggesting sweet sorghum is exclusively suitable for phytoremediation of slight Cd-polluted arable lands. Taken together, enhanced Cd accumulation in shoots of sweet sorghum by excess nitrate application is closely correlated with sulfur metabolism containing elevated ATPS activities, NPT and PCs concentrations in leaves.

Changes in ATP Sulfurylase Activity in Response to Altered Cyanobacteria Growth Conditions

We investigated variations in cell growth and ATP Sulfurylase (ATPS) activity when two cyanobacterial strains—Synechocystis sp. PCC6803 and Synechococcus sp. WH7803—were grown in conventional media, and media with low ammonium, low sulfate and a high CO2/low O2 atmosphere. In both organisms, a transition and adaptation to the reconstructed environmental media resulted in a decrease in ATPS activity. This variation appears to be decoupled from growth rate, suggesting the enzyme is not rate-limiting in S assimilation and raising questions about the role of ATPS redox regulation in cell physiology and throughout Earth history.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman–Birk protease inhibitor and suppresses the accumulation of the β-subunit of β-conglycinin in soybean seeds

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5′-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman–Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC–MS and GC–MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37–52% and 15–19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

ATP sulfurylase activity of sulfate-reducing bacteria from various ecotopes.

Sulfate-reducing bacteria (SRB) are widespread in various ecotopes despite their growth and enzymatic features not compared. In this study, the enzymatic parameters of ATP sulfurylase in cell-free extracts of sulfate-reducing bacteria isolated from various ecotopes such as soils, corrosion products and human large intestine were determined. Comparative analysis of both enzyme characteristics and growth parameters were carried out and similar research has not been reported yet. The initial and maximum rates of enzymatic reaction catalyzed by ATP sulfurylase were significantly different (p < 0.05) in the bacterial strains isolated from various environmental ecotopes. The specific activity of this enzyme in sulfate-reducing bacteria was determined for corrosive and intestinal strains 0.98-1.56 and 0.98-2.26 U × mg-1 protein, respectively. The Michaelis constants were 1.55-2.29mM for corrosive and 2.93-3.13mM for intestinal strains and the affinity range were demonstrated. Based on cluster analysis, the parameters of physiological and biochemical characteristics of sulfate-reducing bacteria from different ecotopes are divided into 3 clusters corresponding to the location of their isolation (soils, heating systems and human intestine). Understanding the enzymatic parameters of the initial stages of sulfate consumption in the process of dissimilatory sulfate reduction will allow the development of effective methods for controlling the production of toxic metabolites, including hydrogen sulfide.

Real-time fluorescence assays of alkaline phosphatase and ATP sulfurylase activities based on a novel PPi fluorescent probe

An anthracene-armed tetraaza macrocyclic fluorescent probe 3-(9-anthrylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(l) for detecting Zn2+ in aqueous medium was synthesized. L–Zn2+ complex, showed selectivity toward pyrophosphate ion (PPi) by quenching the fluorescence in aqueous HEPES buffer (pH 7.4). Furthermore, L–Zn2+ was also used to set up a real-time fluorescence assay for monitoring enzyme activities of alkaline phosphatase (ALP) and adenosine triphosphate sulfurylase (ATPS). In the presence of ALP inhibitor Na3VO4 and ATPS inhibitor chlorate, two enzymes activities decreased obviously, respectively.

Sulfur metabolism: Different tolerances of two aquatic macrophytes exposed to arsenic

The toxicity of arsenic (As) and the mechanisms of response to this pollutant were analyzed in two aquatic plant species, one sensitive and one tolerant to the pollutant, Salvinia minima and Lemna gibba, respectively. The plants, grown in nutrient solution at pH 6.5, were exposed to As concentrations of 0.0 and 1.0mgL−1 for 3 days. Both species accumulated As in their tissues, which resulted in increases in H2O2 production. L. gibba accumulated eleven times more As than S. minima. However, L. gibba was more tolerant, as shown by the absence of cell membrane damage and, despite greater accumulation, smaller growth reduction than S. minima. Indeed, the index of tolerance to As was twenty percent higher in L. gibba than in S. minima, which most likely results from the presence of a more efficient defense system. This defense system in L. gibba is most likely based on sulfate absorption, assimilation and metabolism. L. gibba showed an increase in sulfate absorption and adenosine-5′-triphosphate (ATP) sulfurylase activity (the first enzyme of the inorganic sulfate assimilation pathway) following exposure to As. Consequently, the plant produced greater concentrations of sulfur-containing compounds that are involved in cellular detoxification, such as glutathione and non-protein thiols, and demonstrated greater enzymatic activity of γ-glutamylcysteine synthetase, glutathione S-transferase and glutathione reductase. Therefore, the plant׳s ability to increase absorption, assimilation and metabolism of sulfur are key steps for tolerance to oxidative stress triggered by metals.

유채 품종별 황 공급수준이 황산염 동화에 미치는 영향

유채 품종에서 황 공급수준이 황산염 흡수 및 동화에 미치는 영향을 구명하고자 어린 잎과 성엽 조직에서 ATP sulfurylase (ATPs), <TEX>${SO_4}^{2-}$</TEX> 흡수, 글루타치온 함량을 분석하였다. 본 실험에서 10가지 유채 품종들 (Mosa, Capitol, Saturnin, Akela, Pollen, Mokpo, Youngsan, Tamra Colosse 그리고 Naehan)은 황 공급수준에 따라 몇 가지 황산염 흡수와 동화 능력이 다르게 나타났다. 황 결핍 조건에서 모든 품종의 ATP sulfurylase (ATPs) 활력은 늙은 잎에 비해 어린 잎에서 높게 나타났으며, 글루타치온 함량은 황 공급수준이 감소함에 따라 어린 잎에서 많이 증가하였다. 이러한 결과들은 유채 품종별 황 결핍조건에서 황을 이용하는 능력이 다르다는 것을 잘 보여주었다. To determine sulfate uptake and assimilation of various rape cultivars (Brassica napus L.) under different S levels, the activity of ATP sulfurylase, <TEX>${SO_4}^{2-}$</TEX> uptake and glutathione (GSH) concentrations were measured in different age of leaves. In this study, ten rape cultivars (Mokpo, Tamra, Youngsan, Naehan, Saturnin, Akela, Mosa, Capitol, Pollen, and Colosse) showed various sulfate uptake and assimilation capacity in response to inefficient sulfate supply. Under inadequate sulfate conditions, a greater ATP sulfurylase activity in young leaves was observed in all cultivars compared to that of old leaves. In addition, GSH concentration was considerably increased in young leaves when S supply was declined from 2.0 to 0.2 mM. These results suggested that rape cultivars differ in their capacity to utilize sulfate under limited S conditions.

Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation

Lycopersicon esculantum sulfate transporter gene (LeST 1.1) encodes a high-affinity sulfate transporter (HAST) located in root epidermis. In this study, the LeST 1.1 gene was constitutively expressed in Indian mustard (Brassica juncea cv. Pusa Jai Kisan). Transgenic as well as untransformed plants were grown in sulfur-insufficient (25 and 50 μM) and sulfur-sufficient (1,000 μM) conditions for 30 days. Two-fold increase was noticed in the sulfate uptake rate of transgenic plants grown in both sulfur-insufficient and -sufficient conditions as compared to untransformed plants. The transgenic B. juncea plants were able to accumulate higher biomass and showed improved sulfur status even in sulfur-insufficient conditions when compared with untransformed plants. Chlorophyll content, ATP sulfurylase activity and protein content were also higher in transgenic plants than untranformed plants under sulfur-insufficient conditions. Our results, thus, clearly indicate that constitutive expression of LeST 1.1 gene in B. juncea had led to enhanced capacity of sulfur uptake and assimilation even in sulfur-insufficient conditions. This approach can also be used in other crops to enhance their sulfate uptake and assimilation potential under S-insufficient conditions.

Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity.

The Tbd_0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 A resolution X-ray crystal structure reported here revealed a hexameric assembly with D(3) symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans

The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had <2% APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP + sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

Sulphate uptake and metabolism in water hyacinth and salvinia during cadmium stress

Water hyacinth ( Eichhornia crassipes (Mart.) Solms) and salvinia ( Salvinia auriculata Aubl.) were exposed to toxic levels of Cd with the objective of evaluating its effect on sulphate uptake and metabolism. Plants were treated with 0 and 5 μmol L −1 Cd for 3 days and, then sulphate uptake, ATP sulfurylase activity, soluble thiol content and Cd-binding complexes were determined. Water hyacinth showed a lower sulphate uptake, but its kinetic parameters were not affected by Cd. In salvinia, however, both V max and affinity to sulphate (1/ K m ) decreased with Cd treatment. The ATP sulfurylase activity increased in Cd-treated plant of both species, except in the roots of salvinia. In the presence of Cd water hyacinth always exhibited higher activity of this enzyme. The total soluble thiol content was always higher in water hyacinth. In Cd treated plants it increased in the leaves of water hyacinth, but decreased in salvinia. Cysteine content increased only in water hyacinth leaves, while γ-glutamylcysteine content increased in the two parts of the plants of both species after Cd treatment, especially in water hyacinth. Glutathione contents, on the contrary, after Cd treatment, reduced in both parts of the plants of water hyacinth but only in the leaves of salvinia. The unidentified thiol fraction content increased with Cd treatment in both species, especially in water hyacinth. Root and leaf extracts of both species showed peaks with maxima at A 265/A 280. In treated plants these peaks coincided with Cd content peaks indicating the formation of Cd-binding peptides. It was estimated that in the presence of Cd about 97% of Cd was associated with these complexes and water hyacinth had 28% more Cd-binding peptides than salvinia. Despite its lower sulphate uptake, water hyacinth showed higher rates of sulfur reduction and assimilation into soluble thiols. Possibly, glutathione is used in water hyacinth roots to synthesize hitherto unidentified Cd-binding peptides.

유채의 SO42-흡수 및 동화에 관한 연구

유채의 황 이용성에 대한 영향을 조사하기 위하여 유채 2 품종(cv. Akela, Colosse)을 2.0mM <TEX>${SO_4}^{2-}$</TEX>와 0.2mM <TEX>${SO_4}^{2-}$</TEX>에서 <TEX>${SO_4}^{2-}$</TEX> 흡수, ATP sulfurylase의 활성과 염조직내의 glutathione(GSH) 함량을 측정하였다. 0.2mM <TEX>${SO_4}^{2-}$</TEX>에서 두 품종 모두 2.0mM <TEX>${SO_4}^{2-}$</TEX>에 비해 현저하게 낮은 <TEX>${SO_4}^{2-}$</TEX> 흡수율을 나타냈다. 0.2mM <TEX>${SO_4}^{2-}$</TEX>에서 APT sulfurylase의 활성은 어린잎에서 두 품종 모두 증가하였으나 성숙된 잎에서의 활성은 큰 변화가 얼었다. 0.2mM <TEX>${SO_4}^{2-}$</TEX>에서 glutathione의 함량은 어린잎에서 두 품종 모두 증가하였으나 성숙된 잎에서는 Akela에서만 현저하게 감소하였다. 이러한 결과들은 유채품종간의 <TEX>${SO_4}^{2-}$</TEX> 흡수와 ATP sulfurylase의 특이성 뿐만 아니라, <TEX>${SO_4}^{2-}$</TEX> 흡수가 glutathione과 같은 황을 포함하고 있는 화합물로의 동화와 밀접한 관련이 있음을 나타낸다. To investigate the sulfate utilization efficiency that has been examined in rape (Brassica napus L.) cultivars (cv. Akela and Colosse). During 72h of treatment, in two cultivars, <TEX>${SO_4}^{2-}$</TEX> uptake was significantly higher in complete S condition (2.0 mM <TEX>${SO_4}^{2-}$</TEX>) than that of the S-deficient condition (2.0 mM <TEX>${SO_4}^{2-}$</TEX>). In young leaves, ATP sulfurylase activity increased after S-deficient treatment. However, in old leaves, ATP sulfurylase activity was not significantly changed in Akela. Glutathione concentration in young leaves significantly increased in all cultivars examined under S-deficient condition. The rate of increase in glutathigne concentration caused by S-deficiency treatment was higher in Akela. However, in old leaves, the glutathione concentration in Akela significantly decreased. The results suggest that <TEX>${SO_4}^{2-}$</TEX> uptake and ATP sulfurylase in rape plants were species specific, and that <TEX>${SO_4}^{2-}$</TEX> uptake was highly related to its assimilation into S containing compound such as glutathione.

Effect of sulfur on nitrate reductase and ATP sulfurylase Activities in groundnut (Arachis hypogea L.)

Field experiments were conducted to determine the effect of sulfur (S) and Nitrogen (N) on nitrate reductase (NR) and ATP-sulfurylase activities in groundnut cultivars (Arachis hypogea L. cv. Ambar and Kaushal). Two combinations of S (in kg ha-1): OS (-S) and 20S (+S) were used with 20 kg ha-1 N. The application of S enhanced the NR and ATP-sulfurylase activities in both the cultivars at all the growth stages. The application of S also increased soluble protein and chlorophyll content in the all growth stages of both the cultivars. NR and ATP-sulfurylase activities in the leaves were measured at various growth stages as the two enzymes catalyze the rate limiting steps of the assimilatory pathways of nitrate and sulfate, respectively.

Alteration of Lithium Pharmacology through Manipulation of Phosphoadenosine Phosphate Metabolism

Bisphosphate 3'-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium. Here we report a role for 3'-nucleotidase and its substrate, 3'-phosphoadenosine 5'-phosphate (PAP), in mediating the cellular effects of lithium. Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1. Disruption of the yeast 3'-nucleotidase gene or treatment of cells with lithium results in a >80-fold accumulation of PAP and leads to potent growth inhibition. These data indicate that the accumulation of a 3'-nucleotidase substrate, such as PAP, mediates the toxicity of lithium. To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly down-regulated. Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional down-regulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of these effects were reversed by heterologous expression of human sulfurylase and kinase. Collectively, our data support a role for 3'-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for the treatment of bipolar disorder.

Cloning and use of sC as homologous marker for Aspergillus niger transformation

The sC sequence from Aspergillus niger was cloned and developed into a homologous marker system for genetic transformation. The coding region of the sC gene amplified by PCR from the A. niger genome was provided with Aspergillus nidulans expression signals ( gpdA promoter and trpC terminator). This chimeric construct was used to successfully transform a spontaneous sC − isolate of A. niger to prototrophy. The transformants analyzed by Southern analysis showed integration of multiple copies of the transforming DNA. They also exhibited much higher ATP sulfurylase activity than the wild-type A. niger strain reinforcing the molecular data. This demonstrates the usefulness of the sC niger construct, driven by PgpdA, as a marker for A. niger transformation.

Identification and Functional Characterization of the Novel BM-motif in the Murine Phosphoadenosine Phosphosulfate (PAPS) Synthetase

PAPS synthetase (SK) catalyzes the two sequential reactions of phosphoadenosine phosphosulfate (PAPS) synthesis. A functional motif in the kinase domain of mouse SK, designated the BM-motif ((86) LDGDNhRxhh(N/S)(K/R)(97)), was defined in the course of identifying the brachymorphic (bm) defect. Sequence comparison and the secondary structure predicted for APS kinase suggest that the BM-motif consists of a DGD-turn sequence flanked by other conserved residues. Mutational analysis of the DGD-turn revealed that a flexible and neutral amino acid is preferred at residue 88, that negatively charged residues are strictly required at positions 87 and 89, and that the active site is rigid. The reduction in kinase activity for all DGD-turn mutants, except G88A, was much less severe than the reduction in overall activity, indicating that the BM-motif may also be playing a role in adenosine phosphosulfate (APS) channeling. Two switch mutations, LD86DL and DN89ND, designed to test the positional constraints of Asp(87) and Asp(89), exhibited complete loss of both kinase and overall activities, while LD86DL also exhibited a significant (60%) loss of reverse sulfurylase activity, suggesting that this peptide region is interacting with the sulfurylase domain as well as functioning in the kinase reaction. Other residues targeted for mutational analysis were the highly conserved flanking Asn(90), Arg(92), and Lys(97). N90A resulted in a partial (30%) loss in kinase and overall activities, R92A exhibited total loss of kinase and overall activities, and K97A had no effect on any of the three activities. The complexity of the bifunctional SK in catalyzing the kinase reaction and channeling APS is illustrated by the strict requirements of this novel structural motif in the kinase active site.

Strategies for the allocation of resources under sulfur limitation in the green alga Dunaliella salina.

The effect of sulfur limitation on the partitioning of carbon, nitrogen, and sulfur was investigated in Dunaliella salina. D. salina was able to adapt to 6 microM sulfate; under these conditions, the cells showed reduced growth and photosynthetic rates. Whereas intracellular sulfate was depleted, phosphate, nitrate, and ammonium increased. Amino acids showed a general increase, and alanine became the most abundant amino acid. The activities of four key enzymes of carbon, sulfur, and nitrogen metabolism were differentially regulated: Adenosine 5' triphosphate sulfurylase activity increased 4-fold, nitrate reductase and phosphoenolpyruvate (PEP) carboxylase activities decreased 4- and 11-fold, respectively, whereas carbonic anhydrase activity remained unchanged. Sulfur limitation elicited specific increase or decrease of the abundance of several proteins, such us Rubisco, PEP carboxylase, and a light harvesting complex protein. The accumulation of potentially toxic ammonium indicates an insufficient availability of carbon skeletons. Sulfur deficiency thus induces an imbalance between carbon and nitrogen. The dramatic reduction in PEP carboxylase activity suggests that carbon was diverted away from anaplerosis and possibly channeled into C3 metabolism. These results indicate that it is the coordination of key steps and components of carbon, nitrogen, and sulfur metabolism that allows D. salina to adapt to prolonged sulfur limitation.

Differential subcellular localization and expression of ATP sulfurylase and 5'-adenylylsulfate reductase during ontogenesis of Arabidopsis leaves indicates that cytosolic and plastid forms of ATP sulfurylase may have specialized functions.

ATP sulfurylase and 5'-adenylylsulfate (APS) reductase catalyze two reactions in the sulfate assimilation pathway. Cell fractionation of Arabidopsis leaves revealed that ATP sulfurylase isoenzymes exist in the chloroplast and the cytosol, whereas APS reductase is localized exclusively in chloroplasts. During development of Arabidopsis plants the total activity of ATP sulfurylase and APS reductase declines by 3-fold in leaves. The decline in APS reductase can be attributed to a reduction of enzyme during aging of individual leaves, the highest activity occurring in the youngest leaves and the lowest in fully expanded leaves. By contrast, total ATP sulfurylase activity declines proportionally in all the leaves. The distinct behavior of ATP sulfurylase can be attributed to reciprocal expression of the chloroplast and cytosolic isoenzymes. The chloroplast form, representing the more abundant isoenzyme, declines in parallel with APS reductase during aging; however, the cytosolic form increases over the same period. In total, the results suggest that cytosolic ATP sulfurylase plays a specialized function that is probably unrelated to sulfate reduction. A plausible function could be in generating APS for sulfation reactions.

Functional characterization of a gene encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana.

ATP sulfurylase (ATP: sulfate adenylyl transferase, EC 2.7.7.4), the first enzyme of the sulfate assimilation pathway, is present in the chloroplast and cytosol of plants. In Arabidopsis thaliana cDNA cloning revealed the existence of three ATP sulfurylase isoforms (APS1, -2, and -3) all of which appear to be localized in plastids. In the present study the cytosolic isoform was sought by searching the expressed sequence tag (EST) database and by screening A. thaliana genomic libraries. A fourth isoform, APS4, was identified, but it also encodes a plastid-localized isoform. The APS genes all contain four introns. The introns are located at identical positions within the coding sequence of each of the APS genes. A putative TATA box was identified in the promoter of the APS3 and APS4 genes, but no regions of sequence similarity were found among the other promoters. Combined analysis of an APS4 cDNA and genomic clone revealed that the deduced protein is 469 amino acids and is most homologous to the A. thaliana APS1 subclass. The APS4 cDNA was able to functionally complement a yeast ATP sulfurylase (met3) mutant and the recombinant enzyme displayed ATP sulfurylase activity. The APS4 protein exhibits a plastid targeting peptide at its amino terminus that, when fused to green fluorescent protein, was able to target the reporter to chloroplasts. APS4 mRNA was detected at a similar steady-state level in roots and leaves, and its expression was not induced by sulfur starvation or by O-acetylserine treatment. Having identified a fourth plastid-localized ATP sulfurylase, the origin of cytosolic isoform in A. thaliana remains unclear. Based on sequence analysis, it is hypothesized that APS2 may encode the cytosolic ATP sulfurylase.

Genomic Organization of the Mouse and Human Genes Encoding the ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase Isoform SK2

Mammalian ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase consists of kinase and sulfurylase domains, and catalyzes two sequential reactions to synthesize the universal sulfate donor, phosphoadenosine phosphosulfate (PAPS). In simpler organisms, the ATP sulfurylase and APS kinase reactions are catalyzed by separate enzymes encoded by two or three genes, suggesting that a fusion of separate genes during the course of evolution generated the bifunctional enzyme. We have characterized the genomic structure of the PAPS synthetase SK2 isoform genes for mouse (MSK2) and human (HSK2) and analyzed the possible fusion region. The MSK2 and HSK2 genes exhibit a common structure of 13 exons, including a 15-nucleotide alternatively spliced exon 8. Enzyme activities of several bacterially expressed exon assemblages showed exons 1-6 encode APS kinase, while exons 6-13 encode ATP sulfurylase. The MSK2 construct without the exon 6-encoded peptide showed no kinase or sulfurylase activity, demonstrating that exon 6 encodes sequences required for both activities. Exon 1 and its 5'-flanking sequence are highly divergent between the two species, and intron 1 of the HSK2 gene contains a region similar to the MSK2 promoter sequence, suggesting that it may be the remnant of a now-superceded regulatory region. The HSK2 promoter contains a GC-rich region, not present in the mouse promoter, and has few transcription factor binding sites in common with MSK2. These differences in the two promoter regions suggest that species-specific mechanisms regulate expression of the SK2 isoform.