Phosphoadenosine 5'-phosphosulfate Synthetase Research Articles

Redox switching mechanism of the adenosine 5′-phosphosulfate kinase domain (APSK2) of human PAPS synthase 2

Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the rate-limiting biosynthetic step of the universal sulfuryl donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In higher eukaryotes, the APSK and ATP sulfurylase (ATPS) domains are fused in a single chain. Humans have two bifunctional PAPS synthetase isoforms: PAPSS1 with the APSK1 domain and PAPSS2 containing the APSK2 domain. APSK2 displays a distinct higher activity for PAPSS2-mediated PAPS biosynthesis during tumorigenesis. How APSK2 achieves excess PAPS production has remained unclear. APSK1 and APSK2 lack the conventional redox-regulatory element present in plant PAPSS homologs. Here we elucidate the dynamic substrate recognition mechanism of APSK2. We discover that APSK1 contains a species-specific Cys-Cys redox-regulatory element that APSK2 lacks. The absence of this element in APSK2 enhances its enzymatic activity for excess PAPS production and promotes cancer development. Our results help to understand the roles of human PAPSSs during cell development and may facilitate PAPSS2-specific drug discovery.

Preparation of Tyrosylprotein Sulfotransferases for In Vitro One-Pot Enzymatic Synthesis of Sulfated Proteins/Peptides.

Protein tyrosine sulfation (PTS), catalyzed by membrane-anchored tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications of secretory and transmembrane proteins. PTS, a key modulator of extracellular protein–protein interactions, accounts for various important biological activities, namely, virus entry, inflammation, coagulation, and sterility. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying PTS in biology. A sulfated protein was prepared using a TPST-coupled protein sulfation system that involves the generation of the active sulfate 3′-phosphoadenosine-5′-phosphosulfate (PAPS) through either PAPS synthetase (PAPSS) or phenol sulfotransferase. The preparation of sulfated proteins was confirmed through radiometric or immunochemical assays. In this study, enzymatically active Drosophila melanogaster TPST (DmTPST) and human TPSTs (hTPST1 and hTPST2) were expressed in Escherichia coli BL21(DE3) host cells and purified to homogeneity in high yield. Our results revealed that recombinant DmTPST was particularly useful considering its catalytic efficiency and ease of preparation in large quantities. This study provides tools for high-efficiency, one-step synthesis of sulfated proteins and peptides that are useful for further deciphering the mechanisms, functions, and future applications of PTS.

Structural Mechanism for Substrate Inhibition of the Adenosine 5′-Phosphosulfate Kinase Domain of Human 3′-Phosphoadenosine 5′-Phosphosulfate Synthetase 1 and Its Ramifications for Enzyme Regulation

In mammals, the universal sulfuryl group donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is synthesized in two steps by a bifunctional enzyme called PAPS synthetase. The APS kinase domain of PAPS synthetase catalyzes the second step in which APS, the product of the ATP-sulfurylase domain, is phosphorylated on its 3'-hydroxyl group to yield PAPS. The substrate APS acts as a strong uncompetitive inhibitor of the APS kinase reaction. We generated truncated and point mutants of the APS kinase domain that are active but devoid of substrate inhibition. Structural analysis of these mutant enzymes reveals the intrasubunit rearrangements that occur upon substrate binding. We also observe intersubunit rearrangements in this dimeric enzyme that result in asymmetry between the two monomers. Our work elucidates the structural elements required for the ability of the substrate APS to inhibit the reaction at micromolar concentrations. Because the ATP-sulfurylase domain of PAPS synthetase influences these elements in the APS kinase domain, we propose that this could be a communication mechanism between the two domains of the bifunctional enzyme.

Synthesis of the sulfate donor PAPS in either theDrosophilagermline or somatic follicle cells can support embryonic dorsal-ventral axis formation

The establishment of dorsal-ventral (DV) polarity in the Drosophila embryo depends upon a localized signal that is generated in the perivitelline space of the egg through the action of a serine proteolytic cascade. Spatial regulation of this pathway is determined by the expression of the pipe gene in a subpopulation of ventral follicle cells in the developing egg chamber. The Pipe protein exhibits homology to vertebrate glycosaminoglycan sulfotransferases. In a previous study, we demonstrated that embryonic DV polarity depends upon the sulfotransferase activity of Pipe. Surprisingly, however, our results also indicated that formation of the embryonic DV axis does not require the synthesis of the high-energy sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the follicle cells in which Pipe is presumed to function. Here, we resolve this apparent paradox by demonstrating that dorsalized embryos are only produced by egg chambers in which both germline and follicle cells lack PAPS synthetase activity. Thus, PAPS produced either in the germline or in the follicular epithelium can support the requirement for Pipe sulfotransferase activity in embryonic DV patterning. This finding indicates the existence of a conduit for the movement of PAPS between the germline and the follicle cells, which highlights a previously unappreciated mechanism of soma/germline cooperation affecting pattern formation.

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 γ-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.

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.

Under-sulfation by PAPS synthetase inhibition modulates the expression of ECM molecules during chondrogenesis

Sulfation of proteoglycans is an important post-translational modification in chondrocytes. We previously found that 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthetase-2 levels increased more than 10-fold during mesenchymal cell chondrogenesis. Given that PAPS is the sole sulfur donor, and is produced only by PAPS synthetase in all cells, increased expression of PAPS synthetase-2 should be a prerequisite for increased sulfation activity of chondrocytes. We found that sodium chlorate, a specific inhibitor of PAPS synthetase, inhibited proteoglycan sulfation during chondrogenesis. In contrast, sodium chlorate unexpectedly induced early expression of type II collagen and increased the number of cartilage nodules during chondrogenesis. Inhibition of sulfation also accelerated the down-regulation of N-cadherin and fibronectin during chondrogenesis. These findings suggest that sulfation has an important regulatory role in coordinating the timely expression of extracellular matrix molecules during chondrogenesis, and that under-sulfation may cause the breakdown of this coordination, leading to premature chondrogenesis.

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains.

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.

Expression, purification and crystallization of human 3'-phosphoadenosine-5'-phosphosulfate synthetase 1.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is used to incorporate sulfate into biomolecules. The human PAPS synthetase 1 catalyzes two steps leading from adenosine triphosphate (ATP) and sulfate to PAPS. The ATP sulfurylase domain catalyzes the formation of the intermediate adenosine-5'-phosphosulfate (APS). The APS kinase domain then adds a phosphate group to the 3'-ribose and releases PAPS. In this article, the recombinant expression, purification and crystallization of the full-length protein is described. In Escherichia coli the protein is only partly soluble and copurifies with GroEL. The pure protein migrates as a dimer in gel-filtration chromatography. It is moderately active, forming 25 nmol PAPS per minute per milligram. Crystals grow to 100 x 100 x 300 micro m and diffract to 1.75 A.

Pharmacogenetics of human 3′-phosphoadenosine 5′-phosphosulfate synthetase 1 (PAPSS1): gene resequencing, sequence variation, and functional genomics

3′-Phosphoadenosine 5′-phosphosulfate (PAPS) is the high-energy “sulfate donor” for reactions catalyzed by sulfotransferase (SULT) enzymes. The strict requirement of SULTs for PAPS suggests that PAPS synthesis might influence the rate of sulfate conjugation. In humans, PAPS is synthesized from ATP and SO 4 2− by two isoforms of PAPS synthetase (PAPSS): PAPSS1 and PAPSS2. As a step toward pharmacogenetic studies, we have resequenced the entire coding sequence of the human PAPSS1 gene, including exon–intron splice junctions, using DNA samples from 60 Caucasian-American and 58 African-American subjects. Twenty-one genetic polymorphisms were observed—1 insertion–deletion event and 20 single nucleotide polymorphisms (SNPs)—including two non-synonymous coding SNPs (cSNPs) that altered the following amino acids: Arg333Cys and Glu531Gln. Twelve pairs of these polymorphisms were tightly linked, and a total of twelve unequivocal haplotypes could be identified—two that were common to both ethnic groups and ten that were ethnic-specific. The Arg333Cys polymorphism, with an allele frequency of 2.5%, was observed only in DNA samples from Caucasian subjects. The Glu531Gln polymorphism was rare, with only a single copy of that allele in a DNA sample from an African-American subject. Transient expression in mammalian cells showed that neither of the non-synonymous cSNPs resulted in a change in the basal level of enzyme activity measured under optimal assay conditions. However, the Glu531Gln polymorphism altered the substrate kinetic properties of the enzyme. The Gln531 variant allozyme had a 5-fold higher K m value for SO 4 2− than did the wild-type allozyme and displayed monophasic kinetics for Na 2SO 4. The wild-type allozyme (Glu531) showed biphasic kinetics for that substrate. These observations represent a step toward testing the hypothesis that genetic variation in PAPS synthesis catalyzed by PAPSS1 might alter in vivo sulfate conjugation.

Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells. -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Human 3′-Phosphoadenosine 5′-Phosphosulfate Synthetase 1 (PAPSS1) and PAPSS2: Gene Cloning, Characterization and Chromosomal Localization

Sulfae conjugation is an important pathway in the metabolism of a large number of exogenous and endogenous compounds. These reactions are catalyzed by sulfotransferase (SULT) enzymes that utilize 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS). Two separate PAPSS cDNAs, PAPSS1 and PAPSS2, have been identified in human tissues. We have cloned and characterized the genes for human PAPSS1 and PAPSS2 to make it possible to study the pharmacogenomics of these enzymes. Both genes consisted of 12 exons with virtually identical exon–intron splice junction locations. All splice junctions conformed to the “GT-AG” rule. The total length of PAPSS1 was approximately 108 kb, while that of PAPSS2 was greater than 37 kb. The 5′-flanking region of PAPSS1 did not include a TATA box sequence near the site of transcription initiation, but PAPSS2 had a TATA motif located 21 bp upstream from the site of transcription initiation. Northern blot analysis showed that the major PAPSS1 and PAPSS2 transcripts were approximately 2.7 and 4.2 kb in length, respectively. PAPSS1 mapped to human chromosome band 4q24 while PAPSS2 mapped to 10q22-23 by fluorescence in situ hybridization analysis. Cloning and structural characterization of PAPSS1 and PAPSS2 will make it possible to perform molecular genetic and pharmacogenomic studies of these important enzymes in humans.

Sulfation in high endothelial venules: cloning and expression of the human PAPS synthetase.

High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. Studies with chlorate, a metabolic inhibitor of sulfation, had previously revealed that production of PAPS (3'-phosphoadenosine-5'-phosphosulfate), the high-energy donor of sulfate, is required for sulfation and high-affinity recognition of HEV sialomucins GlyCAM-1 and CD34 by the lymphocyte homing receptor L-selectin. Here, we report the molecular characterization of a novel 2.5 kb human cDNA from MECA-79+ HEV-derived endothelial cells that encodes the target of chlorate, PAPS synthetase, a multifunctional enzyme containing domains for both ATP sulfurylase and adenosine-5'-phosphosulfate kinase. Functional expression of the isolated cDNA in Chinese hamster ovary cells results in high levels of PAPS synthesis, which is abolished by treatment of the transfected cells with chlorate. Northern blot analysis reveals a wide tissue distribution of PAPS synthetase mRNA in the human body, suggesting that human PAPS synthetase may be important for sulfation not only of HEV sialomucins, but also of many other molecules, including mucins such as the P-selectin ligand PSGL-1, proteoglycans, hormones, neurotransmitters, drugs, and xenobiotics.

A multifunctional Urechis caupo protein, PAPS synthetase, has both ATP sulfurylase and APS kinase activities

The synthesis of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) from inorganic sulfate and ATP requires two enzymes, ATP sulfurylase (SUL) and adenosine-5′-phosphosulfate kinase (KIN). In bacteria, fungi, yeast and plants, the two enzymes are present on separate polypeptide chains. We have identified the first animal gene coding for these enzymes. In the marine worm, Urechis caupo (Uc), both SUL and KIN are present on a single polypeptide chain. This protein, which we call PAPS synthetase (PAPSS), is able to complement yeast mutants lacking either enzyme. The Uc PAPSS mRNA is present in oocytes, but is not translated until after fertilization. At least three adult tissues, gut, ceolomocytes and body wall, also contain the mRNA, but at lower concentrations than are found in embryos. Partial sequences of a similar gene from Caenorhabditis elegans (Ce) were detected in a search of the GenBank expressed sequence tag database. Comparison of these Uc and Ce PAPSS sequences with the sequences of cloned genes from non-animal organisms strongly suggests that the animal genes evolved through the fusion of the SUL- and KIN-encoding genes from lower organisms.