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.     

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg₂⁺ and purported to involve residues D₈₇GD₈₉N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D₈₇ and D₈₉ did not hamper ATPS activity however abolished APSK activity severely. Thus, D₈₇GD₈₉N region is required for stabilization of Mg²⁺-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma³²P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg²⁺ and APS of wild type and mutants supports the experimental results.     

Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila.

In sulfur chemolithotrophic bacteria, the enzyme ATP sulfurylase functions to produce ATP and inorganic sulfate from APS and inorganic pyrophosphate, which is the final step in the biological oxidation of hydrogen sulfide to sulfate. The giant tubeworm, Riftia pachyptila, which lives near hydrothermal vents on the ocean floor, harbors a sulfur chemolithotroph as an endosymbiont in its trophosome tissue. This yet-to-be-named bacterium was found to contain high levels of ATP sulfurylase that may provide a substantial fraction of the organisms ATP. We present here, the crystal structure of ATP sulfurylase from this bacterium at 1.7 A resolution. As predicted from sequence homology, the enzyme folds into distinct N-terminal and catalytic domains, but lacks the APS kinase-like C-terminal domain that is present in fungal ATP sulfurylase. The enzyme crystallizes as a dimer with one subunit in the crystallographic asymmetric unit. Many buried solvent molecules mediate subunit contacts at the interface. Despite the high concentration of sulfate needed for crystallization, no ordered sulfate was observed in the sulfate-binding pocket. The structure reveals a mobile loop positioned over the active site. This loop is in a "closed" or "down" position in the reported crystal structures of fungal ATP sulfurylases, which contained bound substrates, but it is in an "open" or "up" position in the ligand-free Riftia symbiont enzyme. Thus, closure of the loop correlates with occupancy of the active site, although the loop itself does not interact directly with bound ligands. Rather, it appears to assist in the orientation of residues that do interact with active-site ligands. Amino acid differences between the mobile loops of the enzymes from sulfate assimilators and sulfur chemolithotrophs may account for the significant kinetic differences between the two classes of ATP sulfurylase.     

Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli

Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases.     

Molecular basis for G protein control of the prokaryotic ATP sulfurylase

Sulfate assimilation is a critical component of both primary and secondary metabolism. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5'-phosphosulfate (APS). Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. To discover the molecular basis of this unusual role for a G protein, we biochemically characterized and solved the X-ray crystal structure of a complex between Pseudomonas syringae ATPS (CysD) and its associated regulatory G protein (CysN). The structure of CysN*D shows the two proteins in tight association; however, the nucleotides bound to each subunit are spatially segregated. We provide evidence that conserved switch motifs in the G domain of CysN allosterically mediate interactions between the nucleotide binding sites. This structure suggests a molecular mechanism by which conserved G domain architecture is used to energetically link GTP turnover to the production of an essential metabolite.     

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.     

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.     

Crystal Structures of the Kinase Domain of the Sulfate-Activating Complex in Mycobacterium tuberculosis

In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5’-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3’-phosphoadenosine-5’-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.     

酿酒酵母ATP-硫酸化酶在大肠杆菌中的表达纯化及其在焦测序中的应用

ATP-硫酸化酶(ATPS,EC2.7.7.4)是一种可逆催化ATP和SO42-反应生成腺嘌呤-5′-磷酸硫酸(APS)和焦磷酸盐(PPi)的酶,已经用于焦测序反应。以酿酒酵母(Saccharomyces cerevisias,CICC1202)基因组DNA为模板,用PCR扩增得到ATPS基因,并克隆到原核表达质粒pET28a( ),得到重组表达质粒pET28a( )-ATPS,在IPTG诱导下,携带pET28a( )-ATPS的大肠杆菌BL21(DE3)表达分子量约为60kD的带有His标签的ATPS酶,经镍亲和层析和超滤两步纯化后,可得到电泳纯级ATPS,比活达5.1×104u/mg,并成功应用于焦测序反应中。     

Channeling in sulfate activating complexes

The synthesis of activated sulfate (adenosine 5'-phosphosulfate, APS) and inorganic pyrophosphate from ATP and SO4 is remarkably unfavorable: K(eq) approximately 10(-8) under presumed, near-physiological conditions. Consequently, ATP sulfurylases, which catalyze APS synthesis, suffer approximately 10(8)-fold losses in catalytic efficiency in the forward (APS-synthesis) versus reverse reaction. Losses of this magnitude place this catalyst at risk of being unable to supply its nutrients to the cell in a timely fashion. ATP sulfurylase domains are often embedded in multifunctional complexes that are capable of also catalyzing the second of two steps in the sulfate activation pathway: the phosphorylation of APS to produce PAPS (3'-phosphoadenosine 5'-phosphosulfate). The colocalization of these activities in a single scaffold suggests that evolution might have worked around the inefficiency problem by fashioning a system capable of transferring APS directly between the active sites of the complex, thereby avoiding the solution-phase energetics. For these reasons, representatives from each of the three types of sulfate activating complex (SAC) [Homo sapiens (type I); Mycobacterium tuberculosis (type II); and Rhodobacter sphaeroides (type III)] were tested for the ability to channel APS. A channeling assay that optically detects solution-phase APS was devised with APS reductase from M. tuberculosis, a previously uncharacterized enzyme. Channeling was not detected in two of the three types of SAC; however, the type III SAC channels with high efficiency. Structural models of type III reveal a 75 A-long channel that interconnects active-site pairs in the complex and that opens and closes in response to occupancy of those sites.     

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.     

The "regulatory" sulfhydryl group of Penicillium chrysogenum ATP sulfurylase. Cooperative ligand binding after SH modification; chemical and thermodynamic properties

ATP sulfurylase from Penicillium chrysogenum is a homohexamer that contains three free sulfhydryl groups/subunit, only one of which (designated SH-1) can be modified by disulfide, maleimide, and halide reagents under nondenaturing conditions. Modification of SH-1 has only a small effect on kcat but causes the [S]0.5 values for MgATP and SO4(2-) (or MoO4(2-) to increase by an order of magnitude. Additionally, the velocity curves become sigmoidal with a Hill coefficient (nH) of about 2 (Renosto, F., Martin, R. L., and Segel, I. H. (1987) J. Biol. Chem. 262, 16279-16288). Direct equilibrium binding measurements confirmed that [32P]MgATP binds to the SH-modified enzyme in a positively cooperative fashion (nH = 2.0) if a sulfate subsite ligand (e.g. FSO3-) is also present. [35S]Adenosine 5'-phosphosulfate (APS) binding to the SH-modified enzyme displayed positive cooperativity (nH = 1.9) in the absence of a PPi subsite ligand. The results indicate that positive cooperativity requires occupancy of the adenylyl and sulfate (but not the pyrophosphate) subsites. [35S]APS binding to the native enzyme displayed negative cooperativity (or binding to at least two classes of sites). Isotope trapping profiles for the single turnover of [35S]APS: (a) confirmed the equilibrium binding curves, (b) indicated that all six sites/hexamer are catalytically active, and (c) showed that APS does not dissociate at a significant rate from E.APS.PPi. The MgPPi concentration dependence of [35S]APS trapping was indicative of MgPPi binding to two classes of sites on both the native and SH-modified enzyme. Inactivation of the native or SH-modified enzyme by phenylglyoxal in the presence of saturating APS was biphasic. The semilog plots suggested that only half of the sites were highly protected. The cumulative data suggest a model in which pairs of sites or subunits can exist in three different states designated HH (both sites have a high APS affinity, as in the native free enzyme), LL (both sites have a low APS affinity as in the SH-modified enzyme), and LH (as in the APS-occupied native or SH-modified enzyme). Thus, the HH----LH transition displays negative cooperativity for APS binding while the LL----LH transition displays positive cooperativity. The relative reactivities of like-paired SH-reactive reagents were in the order: N-phenylmaleimide greater than N-ethylmaleimide; dithionitropyridine greater than dithionitrobenzoate; thiolyte-MQ greater than thiolyte-MB. The log kmod versus pH curve indicates that the pKa of SH-1 is greater than 9.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1

Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.     

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.     

Allosteric and catalytic functions of the PPi-binding motif in the ATP sulfurylase-GTPase system

ATP sulfurylase, from Escherichia coli K-12, catalyzes and couples the Gibbs potentials of two reactions, GTP hydrolysis and activated sulfate (APS, adenosine 5'-phosphosulfate) synthesis. Coupling these potentials requires that the catalytic cycle include reaction stage-dependent conformational changes that gate the activities of the two active sites. These interactions were probed in a mutagenesis study of a highly conserved pyrophosphate-binding motif (SXGXDS), which is located at the APS-forming active site. The motif appears to be unique to the N-type PPi synthetase family, and mutations in it are linked, in other systems, to citrullinemia, an often fatal orphan disease. The conserved sites in the motif were evaluated individually for their ability to activate GTP hydrolysis (which reports interactions among the activator (AMP or Mg2+.PPi), the enzyme, and GTP), to affect the energetic coupling of the two reactions, and to alter the kinetic constants of the adenylyl transfer reaction in the absence of guanine nucleotide. What emerges from this first mutagenic exploration of the PPi motif in any adenylyltransferase is that the residues of the motif participate differently, and in sometimes profoundly important ways, in the different functions of the enzyme.     

Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity.

The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors.     

Crystal structure of adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum

Adenosine 5'-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step conversion of inorganic sulfate to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). This report presents the 2.0 A resolution crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum. The enzyme crystallized as a homodimer with each subunit folded into a classic kinase motif consisting of a twisted, parallel beta-sheet sandwiched between two alpha-helical bundles. The Walker A motif, (32)GLSASGKS(39), formed the predicted P-loop structure. Superposition of the APS kinase active site region onto several other P-loop-containing proteins revealed that the conserved aspartate residue that usually interacts with the Mg(2+) coordination sphere of MgATP is absent in APS kinase. However, upon MgATP binding, a different aspartate, Asp 61, could shift and bind to the Mg(2+). The sequence (156)KAREGVIKEFT(166), which has been suggested to be a (P)APS motif, is located in a highly protease-susceptible loop that is disordered in both subunits of the free enzyme. MgATP or MgADP protects against proteolysis; APS alone has no effect but augments the protection provided by MgADP. The results suggest that the loop lacks a fixed structure until MgATP or MgADP is bound. The subsequent conformational change together with the potential change promoted by the interaction of MgATP with Asp 61 may define the APS binding site. This model is consistent with the obligatory ordered substrate binding sequence (MgATP or MgADP before APS) as established from steady state kinetics and equilibrium binding studies.