Publications

• The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase.

  Li H., Deyrup A., Mensch J.R. Jr., Domowicz M., Konstantinidis A.K., Schwartz N.B.

  Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphor ... >> More

  Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity. << Less

  J. Biol. Chem. 270:29453-29459(1995) [PubMed] [EuropePMC]

• The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding.

  Harjes S., Bayer P., Scheidig A.J.

  The high energy sulfate donor 3'-phosphoadenosine-5-phosphosulfate (PAPS) is used for sulfate conjugation of extracellular matrix, hormones and drugs. Human PAPS synthetase 1 catalyzes two subsequent reactions starting from ATP and sulfate. First the ATP sulfurylase domain forms APS, then the APS ... >> More

  The high energy sulfate donor 3'-phosphoadenosine-5-phosphosulfate (PAPS) is used for sulfate conjugation of extracellular matrix, hormones and drugs. Human PAPS synthetase 1 catalyzes two subsequent reactions starting from ATP and sulfate. First the ATP sulfurylase domain forms APS, then the APS kinase domain phosphorylates the APS intermediate to PAPS. Up to now the interaction between the two enzymatic activities remained elusive, mainly because of missing structural information. Here we present the crystal structure of human PAPSS1 at 1.8 angstroms resolution. The structure reveals a homodimeric, asymmetric complex with the shape of a chair. The two kinase domains adopt different conformational states, with only one being able to bind its two substrates. The asymmetric binding of ADP to the APS kinase is not only observed in the crystal structure, but can also be detected in solution, using an enzymatic assay. These observations strongly indicate structural changes during the reaction cycle. Furthermore crystals soaked with ADP and APS could be prepared and the corresponding structures could be solved. << Less

  J. Mol. Biol. 347:623-635(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Adenosine-5'-triphosphate-sulfurylase from Arabidopsis thaliana and Escherichia coli are functionally equivalent but structurally and kinetically divergent: nucleotide sequence of two adenosine-5'-triphosphate-sulfurylase cDNAs from Arabidopsis thaliana and analysis of a recombinant enzyme.

  Murillo M., Leustek T.

  ATP-sulfurylase, the first enzyme of sulfate assimilation, catalyzes the formation of adenosine-5'-phosphosulfate from ATP and sulfate. Here we report that the higher plant, Arabidopsis thaliana, contains a three-member, expressed gene family encoding plastid localized forms of ATP sulfurylase. Th ... >> More

  ATP-sulfurylase, the first enzyme of sulfate assimilation, catalyzes the formation of adenosine-5'-phosphosulfate from ATP and sulfate. Here we report that the higher plant, Arabidopsis thaliana, contains a three-member, expressed gene family encoding plastid localized forms of ATP sulfurylase. Three cDNAs from A. thaliana, designated APS1, APS2, and APS3, were isolated by their ability to functionally complement a met3 (ATP sulfurylase) mutant strain of Saccharomyces cerevisiae (yeast). The nucleotide sequence of APS1 was reported previously (1). APS2 and APS3, reported here, have 476- and 465-codon open-reading frames encoding 53.6- and 52.0-kDa polypeptides, respectively. The translation products of both clones are highly homologous to APS1 (66 and 86% identity, respectively) over their entire lengths, including amino terminal sequences resembling transit peptides for plastid localization. Both clones are less homologous to MET3 (25 and 30% identity, respectively). Genomic blot analysis of A. thaliana revealed only three genes with homology to the APS cDNAs and RNA blot analysis showed that APS1 is the most highly expressed member of this gene family. The APS polypeptides share homology with ATP-sulfurylases from fungi, a marine worm and a chemoautotrophic bacterium, but, not from Escherichia coli or Rhizobium meliloti. Analysis of recombinant APS3 showed that the protein is structurally and kinetically similar to fungal ATP-sulfurylase, but very different from the E. coli enzyme. The APS3 polypeptide is a homotetramer with specific activities (mumol primary product x mg protein-1 at pH 8.0, 25 degrees C) for 2.9 for APS synthesis, 30.1 for molybdolysis, and 48.7 for ATP synthesis. Despite the sequence, structural, and kinetic differences between higher plant and E. coli ATP-sulfurylases, APS2 and APS3 are able to functionally complement E. coli cysD and cysN (ATP-sulfurylase) mutant strains. << Less

  Arch. Biochem. Biophys. 323:195-204(1995) [PubMed] [EuropePMC]

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

  Ullrich T.C., Blaesse M., Huber R.

  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 cerevisi ... >> More

  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. << Less

  EMBO J. 20:316-329(2001) [PubMed] [EuropePMC]

• Anatomy of an energy-coupling mechanism--the interlocking catalytic cycles of the ATP sulfurylase-GTPase system.

  Sun M., Leyh T.S.

  ATP sulfurylase, from Escherichia coli K-12, conformationally couples the rates and chemical potentials of the two reactions that it catalyzes, GTP hydrolysis and activated sulfate synthesis. The enzyme is rare among such coupling systems in that it links the potentials of small-molecule chemistri ... >> More

  ATP sulfurylase, from Escherichia coli K-12, conformationally couples the rates and chemical potentials of the two reactions that it catalyzes, GTP hydrolysis and activated sulfate synthesis. The enzyme is rare among such coupling systems in that it links the potentials of small-molecule chemistries to one another, rather than to vectorial motion. The pre-steady-state stages of the catalytic cycle of ATP sulfurylase were studied using tools capable of distinguishing between enzyme-bound and solution-phase product for each of the four products of the enzyme. The study reveals that the two chemistries are linked at multiple points in the reaction coordinate. Linking begins with an isomerization prior to chemistry that initiates an ordered cleavage of the beta,gamma and alpha,beta bonds of GTP and ATP, respectively; the rates of these three sequential events increase successively, causing them to appear simultaneous. Linking is again seen in the late stages of the catalytic cycle: product release is ordered with P(i) departing prior to either GDP or PP(i). Release rate constants are determined for each product and used to construct a quantitative model of the mechanism that accurately predicts the behavior of this complex system. << Less

  Biochemistry 44:13941-13948(2005) [PubMed] [EuropePMC]

• Enzymatic mechanisms of phosphate and sulfate transfer.

  Cleland W.W., Hengge A.C.

  Chem Rev 106:3252-3278(2006) [PubMed] [EuropePMC]

• Molecular cloning, expression, and characterization of human bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase and its functional domains.

  Venkatachalam K.V., Akita H., Strott C.A.

  The universal sulfonate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is synthesized by the concerted action of ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase, which in animals are fused into a bifunctional protein. The cDNA for human PAPS synthase (hPAPSS) along with polymera ... >> More

  The universal sulfonate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is synthesized by the concerted action of ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase, which in animals are fused into a bifunctional protein. The cDNA for human PAPS synthase (hPAPSS) along with polymerase chain reaction products corresponding to several NH2- and COOH-terminal fragments were cloned and expressed in COS-1 cells. A 1-268-amino acid fragment expressed APS kinase activity, whereas a 220-623 fragment evinced ATP sulfurylase activity. The 1-268 fragment and full-length hPAPSS (1-623) exhibited hyperbolic responses against APS substrate with equivalent Km values (0.6 and 0.4 microM, respectively). The 1-268 fragment demonstrated Michaelis-Menten kinetics against ATP as substrate (Km 0.26 mM); however, full-length hPAPSS exhibited a sigmoidal response (apparent Km 1.5 mM) suggesting cooperative binding. Catalytic efficiency (Vmax/Km) of the 1-268 fragment was 64-fold higher than full-length hPAPSS for ATP. The kinetic data suggest that the COOH-terminal domain of hPAPSS exerts a regulatory role over APS kinase activity located in the NH2-terminal domain of this bifunctional protein. In addition, the 1-268 fragment and full-length hPAPSS were overexpressed in Escherichia coli and column purified. Purified full-length hPAPSS, in contrast to the COS-1 cell-expressed cDNA construct, exhibited a hyperbolic response curve against ATP suggesting that hPAPSS is perhaps modified in vivo. << Less

  J. Biol. Chem. 273:19311-19320(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Harjes S., Scheidig A., Bayer P.

  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'-phosphos ... >> More

  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. << Less

  Acta Crystallogr. D 60:350-352(2004) [PubMed] [EuropePMC]