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At least 19 recordsLinked to original sources
PubMed1994

On the iron-sulfur cluster of adenosine phosphosulfate reductase from Desulfovibrio vulgaris (Hildenborough).

Adenosine phosphosulfate reductase from Desulfovibrio vulgaris Hildenborough has been purified to homogeneity and was found to consist of two subunits. The alpha and beta subunits have molecular masses of 67.8 kDa and 25.6 kDa, respectively. The apparent molecular mass of the protein is dependent on the ionic strength of the buffer. At low ionic strength, a high molecular-mass multimer is formed, which reversibly changes into smaller units upon addition of salt. The smallest catalytically active unit of the enzyme has a molecular-mass of 186 kDa, as determined by gel-filtration chromatography and, therefore, an alpha 2 beta 2 stoichiometry is proposed. The protein was found to contain 5.6 +/- 1.1 iron and 4.4 +/- 0.6 acid-labile sulfur atoms/alpha beta heterodimer. The reduced protein exhibits a single, rhombic S = 1/2 signal with g values 2.070, 1.932 and 1.891. Lowering the ionic strength of the buffer reversibly changes this spectrum into a complex EPR spectrum, indicating intermolecular, dipolar magnetic coupling. Spin quantification of the reduced protein either at low or at high ionic strength never resulted in more than 1 spin/alpha beta heterodimer. Hence, it follows that the iron and sulfur atoms are arranged in one single cluster. The reduction potential of the iron sulfur cluster, measured in an EPR-monitored redox titration, was found to be -19 mV versus the normal hydrogen electrode (NHE) at pH 7.5. The reduction potential of the flavin measured in an optical titration was found to be -59 mV against NHE at pH 7.5. The flavin behaves as a two-electron-transferring group; no evidence was obtained for a stabilization of the intermediate semiquinone state in the enzyme. Determination of the kinetic parameters of adenosine 5'-phosphosulfate (Ado-PSO4) reductase for its substrates resulted in Km values for sulfite and AMP of 130 microM and 50 microM, respectively. It is proposed that AdoPSO4 reductase contains a single novel Fe/S structure, possibly with an iron-nuclearity greater than four.

Animals↗
PubMed1990

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants : use of adenosine-5'-phosphosulfate and adenosine-3'-phosphate 5'-phosphosulfate as precursors.

Adenosine-5'-phosphosulfate (APS) and adenosine-3'-phosphate 5'-phosphosulfate (PAPS) have been used as precursors of sulfoquinovosyldiacylglycerol (SQDG) in intact chloroplasts incubated in the dark. Competition studies demonstrated APS was preferred over PAPS and SO(4) (2-). Rates of SQDG synthesis up to 3 nanomoles per milligram of chlorophyll per hour were observed when [(35)S]APS and appropriate cofactors were supplied to chloroplasts incubated in the dark. The pH optimum for utilization of APS was 7.0. The incorporation was linear for at least 30 minutes. ATP and UTP stimulated the incorporation of sulfur from APS into SQDG, but the most stimulatory additions were DHAP and glycerol-3-P. The concentration curve for APS showed a maximum at 20 micromolar in the absence of DHAP and 30 micromolar in the presence of DHAP. The optimum concentration of DHAP for conversion of APS into SQDG was 2 millimolar. Rates of synthesis up to 4 nanomoles per milligram of chlorophyll per hour were observed when [(35)S]PAPS was the sulfur donor and appropriate cofactors were supplied to chloroplasts. Optimal rates for conversion of sulfur from PAPS into SQDG occurred with concentrations of DHAP between 5 and 10 millimolar. DHAP was by far the most effective cofactor, although ATP and UTP also stimulated the utilization of PAPS for SQDG biosynthesis. In general, triose phosphates, including glycerol-3-P were not effective cofactors for SQDG biosynthesis.

Journal Article↗
PubMed1989

Synthesis and properties of a nonhydrolyzable adenosine phosphosulfate analog.

Initial activation of inorganic sulfate for subsequent synthesis of sulfated biomolecules requires the action of ATP-sulfurylase to generate adenosine 5'-phosphosulfate (APS). This activated sulfate intermediate is both chemically labile and susceptible to enzymatic degradation. Consequently, it has not proven useful as a ligand for either purification or characterization of the various APS-utilizing enzymes. For these purposes, a stable analog of APS was required. This paper describes the simple and efficient synthesis and structural confirmation of a nonhydrolyzable APS analog, beta-methylene APS, with an overall molar yield of 40-50%. The method involves nucleophilic substitution of the chlorine moiety of a 5'-chloromethylphosphonate ester of 2',3'-O-isopropylidene adenosine by a sulfite ion. We also report the initial utilization of this compound as an inhibitor in kinetic trials of both ATP-sulfurylase and APS kinase and as an affinity ligand for the purification of these two APS-utilizing enzymes from cartilaginous tissue.

Adenosine Monophosphate↗
PubMed1988

Synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA) from adenosine 5'-phosphosulfate and adenosine 5'-triphosphate catalyzed by yeast AppppA phosphorylase.

A novel way of enzymatic synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), which does not involve aminoacyl-tRNA synthetases, has been discovered. Yeast AppppA alpha, beta-phosphorylase catalyzes irreversible conversion of adenosine 5'-phosphosulfate (APS) and ATP into AppppA according to the equation APS + ATP----AppppA + sulfate. In this reaction, the enzyme exhibits a broad pH optimum (between 6 and 8) and requires Mn2+, Mg2+, or Ca2+ ions for activity, with Mn2+ being twice as effective as Mg2+ or Ca2+ at optimal concentration (0.5 mM). The Km values computed for APS and ATP are 80 microM and 700 microM, respectively. The rate constant for the AppppA synthesis is 3 s-1 (pH 8.0, 30 degrees C, 0.5 mM MgCl2). Some ATP analogues like ppppA, GTP, adenosine 5'-(alpha, beta-methylenetriphosphate), and adenosine 5'-(beta, gamma-methylenetriphosphate), but not dATP, UTP, or CTP, are also substrates for AppppA phosphorylase and accept adenylate from APS with the formation of AppppA, AppppG, Appp(CH2)pA, and App(CH2)ppA, respectively. Functional versatility of yeast AppppA phosphorylase may provide a link between metabolism of AppppA on one hand and metabolism of APS and phosphate on the other and raises the possibility of participation of AppppA in regulation of metabolism of APS and/or inorganic phosphate in yeast.

Adenine Nucleotides↗
PubMed1976

Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.

Calcium↗
PubMed2000

Adenosine 5'-phosphosulfate sulfotransferase and adenosine 5'-phosphosulfate reductase are identical enzymes.

Adenosine 5'-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M(r) of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K(m) = 6.5 microM) and not adenosine 3'-phosphate 5'-phosphosulfate as sulfonyl donor. The V(max) of recombinant Lemna APS sulfotransferase (40 micromol min(-1) mg protein(-1)) was about 10 times higher than the previously published V(max) of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO(3)(2-). Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO(3)(2-) was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Amino Acid Sequence↗
PubMed1981

Sulfohydrolytic degradation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and adenosine 5'-phosphosulfate (APS) by enzymes of a nucleotide pyrophosphatase nature.

The sulfohydrolytic activity to degrade active sulfate (3'-phosphoadenosine 5'-phosphosulfate, PAPS) and its precursor, APS (adenosine 5'-phosphosulfate), with a pH optimum at 9.5 was found to be widely distributed in various tissues of rats. In the liver, the activity was located in plasma membranes and endoplasmic reticula. Triton X-100 solubilized rough and smooth endoplasmic reticula gave two peaks of the activity on gel filtration, both of which had nucleotide pyrophosphatase activities, hydrolyzing the pyrophosphate linkages of ATP, NAD, and UDP-Glc, and the phosphodiester linkage of PNTP (p-nitrophenyl-thymidine 5'-monophosphate) besides PAPS and APS.

Adenine Nucleotides↗
PubMed2000

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase.

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.

Adenosine Diphosphate↗
PubMed2002

The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation.

It was generally accepted that plants, algae, and phototrophic bacteria use adenosine 5'-phosphosulfate (APS) for assimilatory sulfate reduction, whereas bacteria and fungi use phosphoadenosine 5'-phosphosulfate (PAPS). The corresponding enzymes, APS and PAPS reductase, share 25-30% identical amino acids. Phylogenetic analysis of APS and PAPS reductase amino acid sequences from different organisms, which were retrieved from the GenBank(TM), revealed two clusters. The first cluster comprised known PAPS reductases from enteric bacteria, cyanobacteria, and yeast. On the other hand, plant APS reductase sequences were clustered together with many bacterial ones, including those from Pseudomonas and Rhizobium. The gene for APS reductase cloned from the APS-reducing cyanobacterium Plectonema also clustered together with the plant sequences, confirming that the two classes of sequences represent PAPS and APS reductases, respectively. Compared with the PAPS reductase, all sequences of the APS reductase cluster contained two additional cysteine pairs homologous to the cysteine residues involved in binding an iron-sulfur cluster in plants. Mössbauer analysis revealed that the recombinant APS reductase from Pseudomonas aeruginosa contains a [4Fe-4S] cluster with the same characteristics as the plant enzyme. We conclude, therefore, that the presence of an iron-sulfur cluster determines the APS specificity of the sulfate-reducing enzymes and thus separates the APS- and PAPS-dependent assimilatory sulfate reduction pathways.

Adenosine Phosphosulfate↗
PubMed1992

Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.

Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.

Adenosine Triphosphate↗
PubMed1999

Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5'-phosphosulfate kinase.

Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 degrees C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t1/2 of 10 min at 37 degrees C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only.

Amino Acid Sequence↗
PubMed1989

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.

Adenosine Monophosphate↗
PubMed2004

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.

Adenosine Phosphosulfate↗
PubMed1998

Colorimetric determination of the purity of 3'-phospho adenosine 5'-phosphosulfate and natural abundance of 3'-phospho adenosine 5'-phosphate at picomole quantities.

This work presents novel colorimetric methods not only to measure 3'-phospho adenosine 5'-phosphate (PAP) and 3'-phospho adenosine 5'-phosphosulfate (PAPS) in the range of picomoles, but also to determine the purity of PAPS or PAP contaminants in PAPS in the range of nanomoles. These methods exploit the availability of overexpressed phenol sulfotransferase (PST) and the fact that sulfuryl group transfer requires the use of PAP or PAPS as a cofactor or cosubstrate. Experimental results indicate that absorption at 400 nm due to the production of 4-nitrophenol (pNP) is catalyzed by PST when the sulfuryl group transfers from 4-nitrophenylsulfate (pNPS) to PAP or to 2-napthol. In the absence of an acceptor substrate, PAPS is hydrolyzed to PAP by PST and is determined by sulfation with pNPS before and after this reaction. The change of absorption of pNP at 400 nm corresponds to the amount of PAP that is hydrolyzed from PAPS. Moreover, a standard curve is constructed using authentic PAP and PAP-free PST. Furthermore, this curve is used to determine the amount of PAP in extracts of pig liver, rat liver, and Escherichia coli.

Adenosine Diphosphate↗
PubMed2002

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.

Adenosine Diphosphate↗
PubMed2001

Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein.

Adenosine 5'-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5'-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minor and Arabidopsis thaliana were overexpressed in Escherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a [4Fe-4S] cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of (57)Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic [4Fe-4S](2+) cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5'-phosphosulfate reductase with a [4Fe-4S] center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5'-phosphosulfate reductases found in sulfate reducing bacteria.

Amino Acid Sequence↗