Crystal structure of adenosine 5'-phosphosulfate kinase isolated from Archaeoglobus fulgidus.

The 3'-phosphoadenosine-5'-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.

Reaction mechanism catalyzed by the dissimilatory adenosine 5'-phosphosulfate reductase. Adenosine 5'-monophosphate inhibitor and key role of arginine 317 in switching the course of catalysis.

The present research is a continuation of our work on dissimilatory reduction pathway of sulfate - involved in biogeochemical sulfur turnover. Adenosine 5'-phosphosulfate reductase (APSR) is the second enzyme in the dissimilatory pathway of the sulfate to sulfide reduction. It reversibly catalyzes formation of the sulfite anion (HSO3-) from adenosine 5'-phosphosulfate (APS) - the activated form of sulfate provided by ATP sulfurylase (ATPS). Two electrons required for this redox reaction derive from reduced FAD cofactor, which is suggested to be involved directly in the catalysis by formation of FADH-SO3- intermediate. The present work covers quantum-mechanical (QM) studies on APSR reaction performed for eight models of APSR active site. The cluster models were constructed based on two crystal structures (PDB codes: 2FJA and 2FJB), differing in conformation of Arg317 active site residue. The described results indicated the most feasible mechanism of APSR forward reaction, including formation of FADHN-SO3- adduct (with proton on N5 atom of isoalloxazine), tautomerization of FADHN-SO3- to FADHO-SO3- (with proton on CO moiety of isoalloxazine), and its reductive cleavage to oxidized FAD and sulfite anion. The reverse reaction proceeds in the backward direction. It is suggested that it requires two AMP molecules, one acting as a substrate and another as an inhibitor of forward reaction, which forces change of Arg317 conformation from "arginine in" (2FJA) to "arginine out" (2FJB). Important role of Arg317 in switching the course of the APSR catalytic reaction is revealed by changing the direction of thermodynamic driving force. The presented research also shows the importance of the protonation pattern of the reduced FAD cofactor and protein residues within the active site.

Adenosine-5'-Phosphosulfate- and Sulfite Reductases Activities of Sulfate-Reducing Bacteria from Various Environments.

A comparative study of the kinetic characteristics (specific activity, initial and maximum rate, and affinity for substrates) of key enzymes of assimilatory sulfate reduction (APS reductase and dissimilatory sulfite reductase) in cell-free extracts of sulphate-reducing bacteria (SRB) from various biotopes was performed. The material for the study represented different strains of SRB from various ecotopes. Microbiological (isolation and cultivation), biochemical (free cell extract preparation) and chemical (enzyme activity determination) methods served in defining kinetic characteristics of SRB enzymes. The determined affinity data for substrates (i.e., sulfite) were 10 times higher for SRB strains isolated from environmental (soil) ecotopes than for strains from the human intestine. The maximum rate of APS reductase reached 0.282-0.862 µmol/min×mg-1 of protein that is only 10 to 28% higher than similar initial values. The maximum rate of sulfite reductase for corrosive relevant collection strains and SRB strains isolated from heating systems were increased by 3 to 10 times. A completely different picture was found for the intestinal SRB Vmax in the strains Desulfovibrio piger Vib-7 (0.67 µmol/min × mg-1 protein) and Desulfomicrobium orale Rod-9 (0.45 µmol/min × mg-1 protein). The determinant in the cluster distribution of SRB strains is the activity of the terminal enzyme of dissimilatory sulfate reduction-sulfite reductase, but not APS reductase. The data obtained from the activity of sulfate reduction enzymes indicated the adaptive plasticity of SRB strains that is manifested in the change in enzymatic activity.

Specific inhibitors of respiratory sulfate reduction: towards a mechanistic understanding.

Microbial sulfate reduction (SR) by sulfate-reducing micro-organisms (SRM) is a primary environmental mechanism of anaerobic organic matter mineralization, and as such influences carbon and sulfur cycling in many natural and engineered environments. In industrial systems, SR results in the generation of hydrogen sulfide, a toxic, corrosive gas with adverse human health effects and significant economic and environmental consequences. Therefore, there has been considerable interest in developing strategies for mitigating hydrogen sulfide production, and several specific inhibitors of SRM have been identified and characterized. Specific inhibitors are compounds that disrupt the metabolism of one group of organisms, with little or no effect on the rest of the community. Putative specific inhibitors of SRM have been used to control sulfidogenesis in industrial and engineered systems. Despite the value of these inhibitors, mechanistic and quantitative studies into the molecular mechanisms of their inhibition have been sparse and unsystematic. The insight garnered by such studies is essential if we are to have a more complete understanding of SR, including the past and current selective pressures acting upon it. Furthermore, the ability to reliably control sulfidogenesis - and potentially assimilatory sulfate pathways - relies on a thorough molecular understanding of inhibition. The scope of this review is to summarize the current state of the field: how we measure and understand inhibition, the targets of specific SR inhibitors and how SRM acclimatize and/or adapt to these stressors.

Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution.

In human pathogens, the sulfate assimilation pathway provides reduced sulfur for biosynthesis of essential metabolites, including cysteine and low-molecular-weight thiol compounds. Sulfonucleotide reductases (SRs) catalyze the first committed step of sulfate reduction. In this reaction, activated sulfate in the form of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is reduced to sulfite. Gene knockout, transcriptomic and proteomic data have established the importance of SRs in oxidative stress-inducible antimicrobial resistance mechanisms. In previous work, we focused on rational and high-throughput design of small-molecule inhibitors that target the active site of SRs. However, another critical goal is to discover functionally important regions in SRs beyond the traditional active site. As an alternative to conservation analysis, we used directed evolution to rapidly identify functional sites in PAPS reductase (PAPR). Four new regions were discovered that are essential to PAPR function and lie outside the substrate binding pocket. Our results highlight the use of directed evolution as a tool to rapidly discover functionally important sites in proteins.

Sulfation pathways in plants.

Plants take up sulfur in the form of sulfate. Sulfate is activated to adenosine 5'-phosphosulfate (APS) and reduced to sulfite and then to sulfide when it is assimilated into amino acid cysteine. Alternatively, APS is phosphorylated to 3'-phosphoadenosine 5'-phosphosulfate (PAPS), and sulfate from PAPS is transferred onto diverse metabolites in its oxidized form. Traditionally, these pathways are referred to as primary and secondary sulfate metabolism, respectively. However, the synthesis of PAPS is essential for plants and even its reduced provision leads to dwarfism. Here the current knowledge of enzymes involved in sulfation pathways of plants will be summarized, the similarities and differences between different kingdoms will be highlighted, and major open questions in the research of plant sulfation will be formulated.

A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent.

Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5'-Phosphosulfate Kinase from Cyanobacteria to Plants.

In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants.

Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20 times and human Hint2 40 times more slowly than human Fhit.

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.

Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation.

Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5'-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3'-phosphoadenosine 5'-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway.

The Saccharomyces cerevisiae gene YPR011c encodes a mitochondrial transporter of adenosine 5'-phosphosulfate and 3'-phospho-adenosine 5'-phosphosulfate.

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier family, nearly all of which have been functionally characterized. In this study, the identification of the mitochondrial carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.

The X-ray crystal structure of APR-B, an atypical adenosine 5'-phosphosulfate reductase from Physcomitrella patens.

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5'-phosphosulfate (APS) reductases possess a cluster and 3'-phosphoadenosine 5'-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.

Structural, biochemical and genetic characterization of dissimilatory ATP sulfurylase from Allochromatium vinosum.

ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO4 (2-)) with ATP to adenosine 5'-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes.

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 PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi 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.

Adenosine-5'-phosphosulfate--a multifaceted modulator of bifunctional 3'-phospho-adenosine-5'-phosphosulfate synthases and related enzymes.

All sulfation reactions rely on active sulfate in the form of 3'-phospho-adenosine-5'-phosphosulfate (PAPS). In fungi, bacteria, and plants, the enzymes responsible for PAPS synthesis, ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase, reside on separate polypeptide chains. In metazoans, however, bifunctional PAPS synthases catalyze the consecutive steps of sulfate activation by converting sulfate to PAPS via the intermediate APS. This intricate molecule and the related nucleotides PAPS and 3'-phospho-adenosine-5'-phosphate modulate the function of various enzymes from sulfation pathways, and these effects are summarized in this review. On the ATP sulfurylase domain that initially produces APS from sulfate and ATP, APS acts as a potent product inhibitor, being competitive with both ATP and sulfate. For the APS kinase domain that phosphorylates APS to PAPS, APS is an uncompetitive substrate inhibitor that can bind both at the ATP/ADP-binding site and the PAPS/APS-binding site. For human PAPS synthase 1, the steady-state concentration of APS has been modelled to be 1.6 µM, but this may increase up to 60 µM under conditions of sulfate excess. It is noteworthy that the APS concentration for maximal APS kinase activity is 15 µM. Finally, we recognized APS as a highly specific stabilizer of bifunctional PAPS synthases. APS most likely stabilizes the APS kinase part of these proteins by forming a dead-end enzyme-ADP-APS complex at APS concentrations between 0.5 and 5 µM; at higher concentrations, APS may bind to the catalytic centers of ATP sulfurylase. Based on the assumption that cellular concentrations of APS fluctuate within this range, APS can therefore be regarded as a key modulator of PAPS synthase functions.

Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances.

Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.

Synthesis of nucleoside phosphosulfates.

We describe an efficient and scalable procedure for the chemical synthesis of nucleoside 5'-phosphosulfates (NPS) from nucleoside 5'-phosphorimidazolides and sulfate bis(tributylammonium) salt. Using this method we obtained various NPS with yields ranging from 70-90%, including adenosine 5'-phosphosulfate (APS) and 2',3'-cyclic precursor of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which are the key intermediates in the assimilation and metabolism of sulfur in all living organisms.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.

Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.