Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.     

ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus

ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus is a bacterial ortholog of the enzyme from filamentous fungi. (The subunit contains an adenosine 5'-phosphosulfate (APS) kinase-like, C-terminal domain.) The enzyme is highly heat stable with a half-life >1h at 90 degrees C. Steady-state kinetics are consistent with a random A-B, ordered P-Q mechanism where A=MgATP, B=SO4(2-), P=PP(i), and Q=APS. The kinetic constants suggest that the enzyme is optimized to act in the direction of ATP+sulfate formation. Chlorate is competitive with sulfate and with APS. In sulfur chemolithotrophs, ATP sulfurylase provides an efficient route for recycling PP(i) produced by biosynthetic reactions. However, the protein possesses low APS kinase activity. Consequently, it may also function to produce PAPS for sulfate ester formation or sulfate assimilation when hydrogen serves as the energy source and a reduced inorganic sulfur source is unavailable.     

Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria. The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents. The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme. The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein. Characterization of the sopT gene has unequivocally shown its bacterial origin. This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium. The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti. This and emerging evidence from other sources suggests that E. coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation. The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.     

The complex structures of ATP sulfurylase with thiosulfate, ADP and chlorate reveal new insights in inhibitory effects and the catalytic cycle.

The ubiquitous enzyme ATP sulfurylase (ATPS) catalyzes the primary step of intracellular sulfate activation, the formation of adenosine 5'-phosphosulfate (APS). It has been shown that the enzyme catalyzes the generation of APS from ATP and inorganic sulfate in vitro and in vivo, and that this reaction can be inhibited by a number of simple molecules. Here, we present the crystal structures of ATPS from the yeast Saccharomyces cerevisiae complexed with compounds that have inhibitory effects on the catalytic reaction of ATPS. Thiosulfate and ADP mimic the substrates sulfate and ATP in the active site, but are non-reactive and thus competitive inhibitors of the sulfurylase reaction. Chlorate is bound in a crevice between the active site and the intermediate domain III of the complex structure. It forms hydrogen bonds to residues of both domains and stabilizes a "closed" conformation, inhibiting the release of the reaction products APS and PPi. These new observations are evidence for the crucial role of the displacement mechanism for the catalysis by ATPS.     

Crystal structure of a novel zinc-binding ATP sulfurylase from Thermus thermophilus HB8

ATP sulfurylase (ATPS) is a ubiquitous enzyme that catalyzes the transfer of the adenylyl group from ATP to inorganic sulfate, producing adenosine 5'-phosphosulfate (APS) and pyrophosphate. The crystal structure of ATPS from Thermus thermophilus HB8 (TtATPS, 347 amino acid residues) in complex with APS was determined at 2.5 A resolution. TtATPS is composed of three domains [domain I (residues 1-134), domain II (residues 135-290), and domain III (residues 291-347)], like the Riftia pachyptila symbiont ATPS, but lacks a fourth domain present in ATPSs from the yeast Saccharomyces cerevisiae and from the fungus Penicillium chrysogenum. TtATPS forms a dimer in the crystal, and the manner of subunit association is different from that observed in dimeric R. pachyptila symbiont ATPS and in the hexameric S. cerevisiae and P. chrysogenum ATPSs. APS is located in the active site of TtATPS, which contains several motifs (QXRN, HXXH, and GRD) conserved in ATPSs. Unexpectedly, TtATPS binds one metal ion per subunit in domain III. XAFS measurement of the crystal and the Bijvoet difference Fourier map unambiguously characterized the metal ion as a zinc ion. The zinc ion is tetrahedrally coordinated by Cys294, Cys297, Cys306, and His310, and could not be removed from the protein by treatment with EDTA. The zinc ion binding site is far from the active site. Because all four residues coordinated to the zinc ion are conserved in the ATPSs from thermophilic bacteria such as Archaeoglobus fulgidus, Pyrococcus abyssi, and Sulfolobus solfataricus, zinc ion chelation may contribute to the thermal stability of these ATPSs.     

Kinetic mechanism of the dimeric ATP sulfurylase from plants

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PP_i (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PP_i in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.     

Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation

ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5'-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family. We have analysed the crystal structure of the native enzyme at 1.95 Angstroms resolution using multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with APS and PPi bound to the active site. The enzyme consists of six identical subunits arranged in two stacked rings in a D:3 symmetric assembly. Nucleotide binding causes significant conformational changes, which lead to a rigid body structural displacement of domains III and IV of the ATPS monomer. Despite having similar folds and active site design, examination of the active site of ATPS and comparison with known structures of related nucleotidylyl transferases reveal a novel ATP binding mode that is peculiar to ATP sulfurylases.     

ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism

Homogeneous ATP sulfurylase from Penicillium chrysogenum has been reported to have an extremely low activity toward its physiological inorganic substrate, sulfate. This low activity is an artifact resulting from potent product inhibition by 5'-adenylylsulfate (APS) (Ki less than 0.25 microM). Assays based on 35S incorporation from 35SO4(2-) into charcoal-adsorbable [35S]APS are nonlinear with time, even in the presence of a large excess of inorganic pyrophosphatase. However, in the presence of excess APS kinase (along with excess pyrophosphatase), the ATP sulfurylase reaction is linear with time and the enzyme has a specific activity (Vmax) of 6 to 7 units mg protein-1 corresponding to an active site turnover number of at least 400 min-1. Monovalent oxyanions such as NO3-, ClO3-, ClO4-, and FSO3- are competitive with sulfate (or molybdate) and essentially uncompetitive with respect to MgATP. However, thiosulfate (SSO3(2-)), a true sulfate analog and dead-end inhibitor of the enzyme (competitive with sulfate or molybdate), exhibited clear noncompetitive inhibition against MgATP. Furthermore, APS was competitive with both MgATP and molybdate in the molybdolysis assay. These results suggest (a) that the mechanism of the normal forward reaction may be random rather than ordered and (b) that the monovalent oxyanions have a much greater affinity for the E X MgATP complex than for free E. In this respect, FSO3-, ClO4-, etc., are not true sulfate analogs although they might mimic an enzyme-bound species formed when MgATP is at the active site. The nonlinear ATP sulfurylase reaction progress curves (with APS accumulating in the presence of excess pyrophosphatase or PPi accumulating in the presence of excess APS kinase) were analyzed by means of "average velocity" plots based on an integrated rate equation. This new approach is useful for enzymes subject to potent product inhibition over a reaction time course in which the substrate concentrations do not change significantly. The analysis showed that ATP sulfurylase has an intrinsic specific activity of 6 to 7 units mg protein-1. Thus, the apparent stimulation of sulfurylase activity by APS kinase results from the continual removal of inhibitory APS rather than from an association of the two sulfate-activating enzymes to form a "3'-phospho-5'-adenylylsulfate synthetase" complex in which the sulfurylase has an increased catalytic activity. The progress curve analyses suggest that APS is competitive with both MgATP and sulfate, while MgPPi is a mixed-type inhibitor with respect to both substrates. The cumulative data point to a random sequence for the forward reaction with APS release being partially rate limiting.     

嗜酸氧化亚铁硫杆菌ATP硫酸化酶的表达、纯化及性质鉴定

ATP硫酸化酶是一种催化ATP和SO42-反应生成腺嘌呤-5’-磷酸硫酸(APS)和焦磷酸盐(PPi)的酶,它是硫酸根同化反应第一步的关键酶。以嗜酸氧化亚铁硫杆菌(A.ferrooxidansATCC 23270)基因组为模板,用PCR扩增得到ATPS基因,并克隆到表达载体pLM1上。加入IPTG的诱导表达,用AKTA蛋白纯化仪的镍柱亲和层析纯化得到浓度和纯度都较高的ATPS蛋白。SDS-PAGE分析,证实其分子量大小为33 kD,并成功的测出了其活性,比活达3.0×103U/mg。     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

Crystal structure of ATP sulfurylase from Penicillium chrysogenum: insights into the allosteric regulation of sulfate assimilation

ATP sulfurylase from Penicillium chrysogenum is an allosterically regulated enzyme composed of six identical 63.7 kDa subunits (573 residues). The C-terminal allosteric domain of each subunit is homologous to APS kinase. In the presence of APS, the enzyme crystallized in the orthorhombic space group (I222) with unit cell parameters of a = 135.7 A, b = 162.1 A, and c = 273.0 A. The X-ray structure at 2.8 A resolution established that the hexameric enzyme is a dimer of triads in the shape of an oblate ellipsoid 140 A diameter x 70 A. Each subunit is divided into a discreet N-terminal domain, a central catalytic domain, and a C-terminal allosteric domain. Two molecules of APS bound per subunit clearly identify the catalytic and allosteric domains. The sequence 197QXRN200 is largely responsible for anchoring the phosphosulfate group of APS at the active site of the catalytic domain. The specificity of the catalytic site for adenine nucleotides is established by specific hydrogen bonds to the protein main chain. APS was bound to the allosteric site through sequence-specific interactions with amino acid side chains that are conserved in true APS kinase. Within a given triad, the allosteric domain of one subunit interacts with the catalytic domain of another. There are also allosteric-allosteric, allosteric-N-terminal, and catalytic-catalytic domain interactions across the triad interface. The overall interactions-each subunit with four others-provide stability to the hexamer as well as a way to propagate a concerted allosteric transition. The structure presented here is believed to be the R state. A solvent channel, 15-70 A wide exists along the 3-fold axis, but substrates have access to the catalytic site only from the external medium. On the other hand, a surface "trench" links each catalytic site in one triad with an allosteric site in the other triad. This trench may be a vestigial feature of a bifunctional ("PAPS synthetase") ancestor of fungal ATP sulfurylase.     

GTPase-mediated activation of ATP sulfurylase.

GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism. The activation of the enzyme, purified from Escherichia coli, is titratable with GTP. The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP. The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis. The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins. This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase.     

Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase.adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no "substrate channeling" of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [35S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a "3'-phosphoadenosine 5'-phosphosulfate synthetase" complex.     

Ligand-induced structural changes in adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum

Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step, ATP-dependent conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS serves as the sulfuryl donor for the biosynthesis of all sulfate esters and also as a precursor of reduced sulfur biomolecules in many organisms. Previously, we determined the crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum [MacRae et al. (2000) Biochemistry 39, 1613-1621]. That structure contained a protease-susceptible disordered region ("mobile lid"; residues 145-170). Addition of MgADP and APS, which together promote the formation of a nonproductive "dead-end" ternary complex, protected the lid from trypsin. This report presents the 1.43 A resolution crystal structure of APS kinase with both ADP and APS bound at the active site and the 2.0 A resolution structure of the enzyme with ADP alone bound. The mobile lid is ordered in both complexes and is shown to provide part of the binding site for APS. That site is formed primarily by the highly conserved Arg 66, Arg 80, and Phe 75 from the protein core and Phe 165 from the mobile lid. The two Phe residues straddle the adenine ring of bound APS. Arg 148, a completely conserved residue, is the only residue in the mobile lid that interacts directly with bound ADP. Ser 34, located in the apex of the P-loop, hydrogen-bonds to the 3'-OH of APS, the phosphoryl transfer target. The structure of the binary E.ADP complex revealed further changes in the active site and N-terminal helix that occur upon the binding/release of (P)APS.     

Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately ) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.     

Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb).     

Cloning of two cDNAs encoding a family of ATP sulfurylase from Camellia sinensis related to selenium or sulfur metabolism and functional expression in Escherichia coli.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of two cDNAs encoding ATP sulfurylase (APS1 and APS2) from Camellia sinensis. They were isolated by RT-PCR and RACE-PCR reactions. The expression of APS1 and APS2 are correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1415-bp cDNA with an open reading frame predicted to encode a 360-amino acid, 40.5kD protein; APS2 is a 1706-bp cDNA with an open reading frame to encode a 465-amino acid, 51.8kD protein. The predicted amino acid sequences of APS1 and APS2 have high similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum, with 86% and 84% identity respectively. However, they share only 59.6% identity with each other. The enzyme extracts prepared from recombinant Escherichia coli containing Camellia sinensis APS genes had significant enzyme activity.     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Control of sulfate concentration by miR395-targeted APS genes in Arabidopsis thaliana

Sulfur nutrition is crucial for plant growth and development,as well as crop yield and quality.Inorganic sulfate in the soil is the major sulfur source for plants.After uptake,sulfate is activated by ATP sulfurylase,and then gets assimilated into sulfur-containing metabolites.However,the mechanism of regulation of sulfate levels by ATP sulfurylase is unclear.Here,we investigated the control of sulfate levels by miR395-mediated regulation of APS1/3/4.Sulfate was over-accumulated in the shoots of miR395 over-expression plants in which the expression of the APS1,APS3,and APS4 genes was suppressed.Accordingly,reduced expression of miR395 caused a decline of sulfate concentration.In agreement with these results,over-expression of the APS1,APS3,and APS4 genes led to the reduction of sulfate levels.Differential expression of these three APS genes in response to sulfate starvation implied that they have different functions.Further investigation revealed that the regulation of sulfate levels mediated by miR395 depends on the repression of its APS targets.Unlike the APS1,APS3,and APS4 genes,which encode plastid-localized ATP sulfurylases,the APS2 gene encodes a cytosolic version of ATP sulfurylase.Genetic analysis indicated that APS2 has no significant effect on sulfate levels.Our data suggest that miR395-targeted APS genes are key regulators of sulfate concentration in leaves.     

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.