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Sulfation is an essential modification on biomolecules in living cells, and 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is its unique and universal sulfate donor. Human PAPS synthases (PAPSS1 and 2) are the only enzymes that catalyze PAPS production from inorganic sulfate. Unexpectedly, PAPSS1 and PAPSS2 do not functional complement with each other, and abnormal function of PAPSS2 but not PAPSS1 leads to numerous human diseases including bone development diseases, hormone disorder and cancers. Here, we reported the crystal structures of ATP-sulfurylase domain of human PAPSS2 (ATPS2) and ATPS2 in complex with is product 5'-phosphosulfate (APS). We demonstrated that ATPS2 recognizes the substrates by using family conserved residues located on the HXXH and PP motifs, and achieves substrate binding and releasing by employing a non-conserved phenylalanine (Phe550) through a never observed flipping mechanism. Our discovery provides additional information to better understand the biological function of PAPSS2 especially in tumorigenesis, and may facilitate the drug discovery against this enzyme.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Phenolic acids are known flavonoid metabolites, which typically undergo bioconjugation during phase II of biotransformation, forming sulfates, along with other conjugates. Sulfated derivatives of phenolic acids can be synthesized by two approaches: chemoenzymatically by 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfotransferases or PAPS-independent aryl sulfotransferases such as those from Desulfitobacterium hafniense, or chemically using SO3 complexes. Both approaches were tested with six selected phenolic acids (2-hydroxyphenylacetic acid (2-HPA), 3-hydroxyphenylacetic acid (3-HPA), 4-hydroxyphenylacetic acid (4-HPA), 3,4-dihydroxyphenylacetic acid (DHPA), 3-(4-hydroxyphenyl)propionic acid (4-HPP), and 3,4-dihydroxyphenylpropionic acid (DHPP)) to create a library of sulfated metabolites of phenolic acids. The sulfates of 3-HPA, 4-HPA, 4-HPP, DHPA, and DHPP were all obtained by the methods of chemical synthesis. In contrast, the enzymatic sulfation of monohydroxyphenolic acids failed probably due to enzyme inhibition, whereas the same reaction was successful for dihydroxyphenolic acids (DHPA and DHPP). Special attention was also paid to the counterions of the sulfates, a topic often poorly reported in synthetic works. The products obtained will serve as authentic analytical standards in metabolic studies and to determine their biological activity.

The synthesis of sulfated polysaccharides involves the sulfation of simpler polysaccharide substrates, through the action sulfotransferases using the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Three enzymes are essential for the in vitro synthesis of PAPS, namely, pyrophosphatase (PPA), adenosine 5'-phosphosulfate kinase (APSK), and ATP sulfurylase (ATPS). The optimized enzyme expression ratio and effect on PAPS synthesis were evaluated using ePathBrick, a novel synthetic biology tool that assemble multiple genes in a single vector. The introduction of multiple promoters and stop codons at different location enable the bacterial system to fine tune expression level of the genes inserted. Recombinant vectors expressing PPA (U39393.1), ATPS (CP021243.1), and PPA (CP047127.1) were used for fermentations and resulted in volumetric yields of 400-1380 mg/L with accumulation of 34-66% in the soluble fraction. The enzymes from soluble fraction, without any further purification, were used for PAPS synthesis. The PAPS was used for the chemoenzymatic synthesis of a heparan sulfate polysaccharide and coupled with a PAPS-ASTIV regeneration system. ASTIV catalyzes the regeneration of PAPS. A recombinant vector expressing the enzyme ASTIV (from Rattus norvegicus) was used for fermentations and resulted in volumetric yield of 1153 mg/L enzyme with accumulation of 48% in the soluble fraction. In conclusion, we have successfully utilized a metabolic engineering approach to optimize the overall PAPS synthesis productivity. In addition, we have demonstrated that the ePathBrick system could be applied towards study and improvement of enzymatic synthesis conditions. In parallel, we have successfully demonstrated an autoinduction microbial fermentation towards the production of mammalian enzyme (ASTIV). KEY POINTS : • ePathBrick used to optimize expression levels of enzymes. • Protocols have been used for the production of recombinant enzymes. • High cell density fed-batch fermentations with high yields of soluble enzymes. • Robust fermentation protocol successfully transferred to contract manufacturing and research facilities.

Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO

Heparin, a glycosaminoglycan-based anticoagulant drug, is prepared as an extract of animal tissues. Heparosan, an Escherichia coli (E. coli) K5 capsular polysaccharide with the structure →4)-β-D-glucuronic acid (1 → 4)-β-D-N-acetylglucosamine (1→, corresponds to the precursor backbone in the Golgi-based biosynthesis of heparin. Anticoagulant heparin is prepared in a one-pot synthesis using a chemically prepared derivative of heparosan called N-sulfoheparosan (NSH), recombinant Golgi enzymes expressed in E. coli, and the 3-phosphoadenosine-5-phosphosulfate (PAPS) cofactor.

3'-Phosphoadenosine 5'-phosphosulfate transporters (PAPSTs) play an important role in transporting 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the universal sulfuryl donor for sulfation, from the cytosol into the lumen of the Golgi apparatus. Here, we describe three methods for the analysis of PAPST; a transporter activity assay with yeast or mammalian cell fraction, real-time reverse transcription polymerase chain reaction on tissue samples, and immunohistochemistry on brain sections.

Sulfotransferases (SULTs) are phase II drug-metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a substrate. It has been previously suggested that a considerable shift of SULT structure caused by PAPS binding could control the capability of SULT to bind large substrates. We employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited normal modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. MDeNM allowed exploring an extended conformational space of PAPS-bound SULT1A1, which has not been achieved up to now by using classical MD. The generated ensembles combined with docking of 132 SULT1A1 ligands shed new light on substrate and inhibitor binding mechanisms. Unexpectedly, our simulations and analyses on binding of the substrates estradiol and fulvestrant demonstrated that large conformational changes of the PAPS-bound SULT1A1 could occur independently of the co-factor movements that could be sufficient to accommodate large substrates as fulvestrant. Such structural displacements detected by the MDeNM simulations in the presence of the co-factor suggest that a wider range of drugs could be recognized by PAPS-bound SULT1A1 and highlight the utility of including MDeNM in protein–ligand interactions studies where major rearrangements are expected.

Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic organic chemistry. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogues have been synthesised that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the production of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogues, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.

Iron (Fe) is an essential micronutrient for plants and is present abundantly in the Earth's crust. However, Fe bioavailability in alkaline soils is low due to the decreased solubility of the ferric ions. Previously, we have demonstrated the relationship between the PAP/SAL1 retrograde signaling pathway, the activity of Strategy I Fe uptake genes (FIT, FRO2, IRT1), and ethylene signaling. In this work, we have characterized mutant lines that are deficient in this retrograde signaling pathway and their ability to grow in alkaline soils. This adverse growth condition caused less impact on mutant plants, which showed less reduced rosette area, and higher carotenoid, chlorophyll and Fe content than wild-type plants. Several genes involved in the biosynthesis and excretion of secondary metabolites derived from the phenylpropanoid pathway, which improve Fe uptake, were elevated in mutant plants. Finally, we observed an increase in excreted fluorescent phenolic compounds in mutant lines compared to wild-type plants. In this way, PAP/SAL1 mutants showed alterations in the biosynthesis of metabolites that mobilize Fe, which ultimately improved these plants ability to grow in alkaline soils. Results agree with the existence of a link between the PAP/SAL1 retrograde signaling pathway and the regulation of Fe deficiency responses in Arabidopsis.

Sulfation is a common modification of extracellular glycans, tyrosine residues on proteins, and steroid hormones, and is important in a wide variety of signaling pathways. We investigated the role of sulfation on endogenous oxidative stress, such as glutamate-induced oxytosis and erastin-induced ferroptosis, using mouse hippocampal HT22 cells. Sodium chlorate competitively inhibits the formation of 3&prime, phosphoadenosine 5&prime, phosphosulfate, the high energy sulfate donor in cellular sulfation reactions. The treatment of HT22 cells with sodium chlorate decreased sulfation of heparan sulfate proteoglycans and chondroitin sulfate proteoglycans. Sodium chlorate and &beta, d-xyloside, which prevents proteoglycan glycosaminoglycan chain attachment, exacerbated both glutamate- and erastin-induced cell death, suggesting that extracellular matrix influenced oxytosis and ferroptosis. Moreover, sodium chlorate enhanced the generation of reactive oxygen species and influx of extracellular Ca2+ in the process of oxytosis and ferroptosis. Interestingly, sodium chlorate did not affect antioxidant glutathione levels. Western blot analysis revealed that sodium chlorate enhanced erastin-induced c-Jun N-terminal kinase phosphorylation, which is preferentially activated by cell stress-inducing signals. Collectively, our findings indicate that sulfation is an important modification for neuroprotection against oxytosis and ferroptosis in neuronal hippocampal cells.

3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a commonly used anticoagulant drug. Previously, using ATP as the substrate, we had developed a one-pot synthesis to prepare PAPS with 47% ATP conversion efficiency. During the reaction, 47% of ATP was converted into the by-product, ADP. Here, to increase the conversion ratio of ATP to PAPS, an ATP regeneration system was developed to couple with PAPS synthesis. In the ATP regeneration system, the chemical compound, monopotassium phosphoenolpyruvate (PEP−K+), was synthesized and used as the phospho-donor. By using 3-bromopyruvic acid as the starting material, the total yield of PEP−K+ synthesis was over 50% at low cost. Then, the enzyme PykA from Escherichia coli was overexpressed, purified, and used to convert the by-product ADP into ATP. When coupled the ATP regeneration system with PAPS synthesis, the higher ratio of PEP−K+ to ADP was associated with higher ATP conversion efficiency. By using the ATP regeneration system, the conversion ratio of ATP to PAPS was increased to 98% as determined by PAMN-HPLC analysis, and 5 g of PAPS was produced in 1 L of the reaction mixture. Furthermore, the chemoenzymatic synthesized PAPS was purified and freeze-dried without observed decomposition. However, the powdery PAPS was more unstable than the PAPS sodium salt in aqueous solution at ambient temperature. This developed chemoenzymatic approach of PAPS production will contribute to the synthesis of heparin, in which PAPS is necessary as the individual sulfo-donor.

Pyrones comprise a structurally diverse class of compounds. Although they are widespread in nature, their specific physiological functions remain unknown in most cases. We recently described that triketide pyrones mediate the sulfotransfer in caprazamycin biosynthesis. Herein, we report the identification of conexipyrones A-C, three previously unrecognized tetra-substituted α-pyrones, from the soil actinobacterium

Sulfated compounds are widely present in cytoplasm, on cell surface, and in extracellular matrix. These compounds play important roles in cell development, differentiation, immune response, detoxication, and cell signal transduction. 3-Phosphoadenosine-5-phosphosulfate (PAPS) is the universal sulfate group donor for the biosynthesis of sulfated compounds. Up to now, the synthesis of PAPS is still too expensive for industrial applications. This review focuses on the recent progress of PAPS production and summaries the application of PAPS, particularly in the production of glucosinolate, heparin, condroitin sulfate, and oxamniquine production.硫酸化化合物广泛存在于胞浆、细胞表面及胞外基质中，在机体细胞发育、分化、免疫、解毒和信号传递等生命活动过程中起着不可替代的作用。3-磷酸腺苷-5-磷酸硫酸 (3-phosphoadenosine-5-phosphosulfate，PAPS)是化合物硫酸化过程中最常用的硫酸基供体，但目前合成PAPS 并最终实现其工业化应用还困难重重。文中主要综述过去10 年内关于PAPS 的生物合成及应用的研究进展，以期为PAPS 的合成及其在芥子油苷、肝素、硫酸软骨素及羟胺硝喹等的生物合成中的应用提供参考。.

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is a key player in the sulfation of biomolecules, but methods for selective measurements are lacking. A liquid chromatography–mass spectrometry (LC–MS) approach for measuring PAPS was developed. A central feature of the method was employing hydrophilic interaction liquid chromatography (HILIC), which is highly suited for separating very polar/charged compounds, and is compatible with electrospray MS. Using simple instrumentation, the analysis time per sample was below 10 min and the method was characterized by easy sample preparation. The method was used to monitor decreasing levels of PAPS as function of sodium chlorate treatment (an inhibitor of PAPS synthesis) in whole-cell lysates as well as Golgi-fractions. The method allowed PAPS to be chromatographically separated from ADP and ATP, which can interfere with measurements if a less resolving LC–MS method is used.

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.

The second messenger c-di-GMP is a key regulator of bacterial physiology. The V. vulnificus genome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation in V. vulnificus. Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers brp-mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen., Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogen Vibrio vulnificus. V. vulnificus, as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of the brp extracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-induced PbrpA expression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), was required for calcium-induced PbrpA expression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger in V. vulnificus. Moreover, c-di-GMP and BrpT (the activator of brp expression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boost brp expression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation of V. vulnificus biofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism.

Sterol sulfates are widely occurring molecules in marine organisms. Their importance has been so far underestimated although many of these compounds are crucial mediators of physiological and ecological functions in other organisms. Biosynthesis of sterol sulfates is controlled by cytosolic sulfotransferases (SULTs), a varied family of enzymes that catalyze the transfer of a sulfo residue (-SO

The principal dietary sources of sulfur, the amino acids methionine and cysteine, may not always be consumed in adequate amounts to meet sulfur requirements. The naturally occurring organosulfur compound, methylsulfonylmethane (MSM), is available as a dietary supplement and has been associated with multiple health benefits. Absorption of MSM by the small intestine and accumulation of the associated sulfur moiety in selected tissues with chronic (8 days) administration were evaluated using juvenile male mice. Intestinal absorption was not saturated at 50 mmol, appeared passive and carrier-independent, with a high capacity (at least 2 g/d-mouse). The 35S associated with MSM did not increase in serum or tissue homogenates between days 2 and 8, indicating a stable equilibrium between intake and elimination was established. In contrast, proteins isolated from the preparations using gel electrophoresis revealed increasing incorporation of 35S in the protein fraction of serum, cellular elements of blood, liver, and small intestine but not skeletal muscle. The potential contributions of protein synthesis using labeled sulfur amino acids synthesized by the gut bacteria and posttranslational sulfation of proteins by incorporation of the labeled sulfate of MSM in 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and subsequent transfer by sulfotransferases are discussed.

In many organisms, 3'-phosphoadenosine 5'-phosphate (PAP) is a product of two reactions in the sulfur activation pathway. The sulfurylation of biomolecules, catalyzed by sulfotransferases, uses 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor, producing the sulfated biomolecule and PAP product. Additionally, the first step in sulfate reduction for many bacteria and fungi reduces the sulfate moiety of PAPS, producing PAP and sulfite, which is subsequently reduced to sulfide. PAP is removed by the phosphatase activity of CysQ, a 3',5'-bisphosphate nucleotidase, yielding AMP and phosphate. Because excess PAP alters the equilibrium of the sulfur pathway and inhibits sulfotransferases, PAP concentrations can affect the levels of sulfur-containing metabolites. Therefore, CysQ, a divalent cation metal-dependent phosphatase, is a major regulator of this pathway. CysQ (Rv2131c) from Mycobacterium tuberculosis (Mtb) was successfully expressed, purified, and crystallized in a variety of ligand-bound states. Here we report six crystal structures of Mtb CysQ, including a ligand-free structure, a lithium-inhibited state with substrate PAP bound, and a product-bound complex with AMP, phosphate, and three Mg(2+) ions bound. Comparison of these structures together with homologues of the superfamily has provided insight into substrate specificity, metal coordination, and catalytic mechanism.

Entamoeba histolytica , a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3′-phosphoadenosine 5′-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba , PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.

Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of small molecules-metabolites, drugs, and other xenobiotics-via the transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. SULT1A1 is the most abundant SULT in liver and has the broadest substrate spectrum of any SULT. Here we present the discovery of a new form of SULT1A1 allosteric regulation that modulates the catalytic efficiency of the enzyme over a 130-fold dynamic range. The molecular basis of the regulation is explored in detail and is shown to be rooted in an energetic coupling between the active-site caps of adjacent subunits in the SULT1A1 dimer. The first nucleotide to bind causes closure of the cap to which it is bound and at the same time stabilizes the cap in the adjacent subunit in the open position. Binding of the second nucleotide causes both caps to open. Cap closure sterically controls active-site access of the nucleotide and acceptor; consequently, the structural changes in the cap that occur as a function of nucleotide occupancy lead to changes in the substrate affinities and turnover of the enzyme. PAPS levels in tissues from a variety of organs suggest that the catalytic efficiency of the enzyme varies across tissues over the full 130-fold range and that efficiency is greatest in those tissues that experience the greatest xenobiotic "load".

In bacteria, 3',5'-adenosine bisphosphate (pAp) is generated from 3'-phosphoadenosine 5'-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5'-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn(2+) ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s(-1) and 1.4 × 10(6) M(-1) s(-1), respectively. The enzyme is promiscuous and is able to hydrolyze other 3',5'-bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2'-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes.

Sulfur is an essential micronutrient involved in diverse cellular functions ranging from the control of intracellular redox states to electron transport. Eukaryotes incorporate sulfur by metabolizing inorganic sulfate into the universal sulfur donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Sulfotransferases then catalyze the donation of the activated sulfur from PAPS to a broad range of acceptors including xenobiotic small molecules and extracellular proteoglycans while also generating the byproduct 3'-phosphoadenosine 5'-phosphate (PAP). In mammals, PAP is regulated by two related 3'-nucleotidases, Golgi-resident PAP phosphatase (gPAPP) and cytoplasmic bisphosphate 3'-nucleotidase 1 (Bpnt1), which hydrolyze PAP to 5'-AMP and whose inactivation results in severe physiological defects. Loss of Bpnt1 in mice leads to the accumulation of PAP in the liver, aberrant nucleolar architecture, and liver failure, all of which can be rescued by genetically repressing PAPS synthesis. Yet interestingly, Bpnt1 protein is expressed at high levels in a majority of tissues, suggesting that additional tissues might also be affected. To investigate this possibility, we closely examined the expression of Bpnt1 protein, accumulation of PAP, and appearance of dysmorphic nucleoli in wild-type and Bpnt1(-/-) mice. Surprisingly, we found that while Bpnt1 protein is widely expressed, only the liver, duodenum, and kidneys contain high levels of PAP and nucleolar reorganization. We hypothesize that these tissues share commonalities such as being highly polarized and situated at the interfaces of fluid reservoirs that might enhance their susceptibility to loss of Bpnt1. These studies highlight the importance of PAP metabolism in extrahepatic tissues and provide a framework for future investigations into the function of Bpnt1 in the kidney and small intestine.

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one-step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least-explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000-fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin-4-sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4-fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS-accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one-step microbial synthesis of CS. This will provide a new platform to produce CS.

Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C5-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin.

The cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of nucleophilic substrates, and the cofactor for sulfonation, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), is biosynthesized from sulfate and ATP. The phenotype of male knockout mice for the NaS1 sodium sulfate cotransporter includes hyposulfatemia and increased hepatic expression of mouse cytoplasmic sulfotransferase Sult2a and Sult3a1. Here we report that in 8-week-old female NaS1-null mice, hepatic Sult2a1 mRNA levels were ∼51-fold higher than they were in a wild-type liver but expression of no other Sult was affected. To address whether hyposulfatemia-inducible Sult2a1 expression might be due to reduced PAPS levels, we stably knocked down PAPS synthases 1 and 2 in HepG2 cells (shPAPSS1/2 cells). When a reporter plasmid containing at least 233 nucleotides (nt) of Sult2a1 5′-flanking sequence was transfected into shPAPSS1/2 cells, reporter activity was significantly increased relative to the activity that was seen for reporters containing 179 or fewer nucleotides. Mutation of an IR0 (inverted repeat of AGGTCA, with 0 intervening bases) nuclear receptor motif at nt −191 to 180 significantly attenuated the PAPSS1/2 knockdown-mediated increase. PAPSS1/2 knockdown significantly activated farnesoid X receptor (FXR), retinoid-related orphan receptor, and pregnane X receptor responsive reporters, and treatment with the FXR agonist GW4064 [3-(2,6-dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole] increased Sult2a1 promoter activity when the IR0 was intact. Transfection of shPAPSS1/2 cells with FXR small interfering RNA (siRNA) significantly reduced the Sult2a1 promoter activity. The impact of PAPSS1/2 knockdown on Sult2a1 promoter activity was recapitulated by knocking down endogenous SULT2A1 expression in HepG2 cells. We propose that hyposulfatemia leads to hepatic PAPS depletion, which causes loss of SULT2A1 activity and results in accumulation of nonsulfated bile acids and FXR activation.

The human cytosolic sulfotransferases (SULTs) comprise a 13-member enzyme family that regulates the activities of hundreds, perhaps thousands, of signaling small molecules via regiospecific transfer of the sulfuryl moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the hydroxyls and amines of acceptors. Signaling molecules regulated by sulfonation include numerous steroid and thyroid hormones, epinephrine, serotonin, and dopamine. SULT1A1, a major phase II metabolism SULT isoform, is found at a high concentration in liver and has recently been show to harbor two allosteric binding sites, each of which binds a separate and complex class of compounds: the catechins (naturally occurring polyphenols) and nonsteroidal anti-inflammatory drugs. Among catechins, epigallocatechin gallate (EGCG) displays high affinity and specificity for SULT1A1. The allosteric network associated with either site has yet to be defined. Here, using equilibrium binding and pre-steady state studies, the network is shown to involve 14 distinct complexes. ECGG binds both the allosteric site and, relatively weakly, the active site of SULT1A1. It is not a SULT1A1 substrate but is sulfonated by SULT2A1. EGCG binds 17-fold more tightly when the active-site cap of the enzyme is closed by the binding of the nucleotide. When nucleotide is saturating, EGCG binds in two phases. In the first, it binds to the cap-open conformer; in the second, it traps the cap in the closed configuration. Cap closure encapsulates the nucleotide, preventing its release; hence, the EGCG-induced cap stabilization slows nucleotide release, inhibiting turnover. Finally, a comprehensive quantitative model of the network is presented.

The sulfuryl transfer reaction is of fundamental biological importance. One of the most important manifestations of this process are the reactions catalyzed by members of the cytosolic sulfotransferase (SULT) superfamily. These enzymes transfer the sulfuryl moiety from the universal donor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a wide variety of substrates with hydroxyl- or amino-groups. Normally a detoxification reaction this facilitates the elimination of a multitude of xenobiotics, although for some molecules sulfation is a bioactivation step. In addition, sulfation plays a key role in endocrine and other signalling pathways since many steroids, sterols, thyroid hormones and catecholamines exist primarily as sulfate conjugates in humans. This article summarizes much of our current knowledge of the organization and function of the human cytosolic sulfotransferases and highlights some of the important interspecies differences that have implications for, among other things, drug development and chemical safety analysis.

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.

Human SULT2A1 is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (-SO(3)) from activated sulfate (PAPS, 3'-phosphoadenosine 5-phosphosulfate) to hundreds of acceptors (metabolites and xenobiotics). Sulfation recodes the biologic activity of acceptors by altering their receptor interactions. The molecular basis on which these enzymes select and sulfonate specific acceptors from complex mixtures of competitors in vivo is a long-standing issue in the SULT field. Raloxifene, a synthetic steroid used in the prevention of osteoporosis, and dehydroepiandrosterone (DHEA), a ubiquitous steroid precusor, are reported to be sulfated efficiently by SULT2A1 in vitro, yet unlike DHEA, raloxifene is not sulfated in vivo. This selectivity was explored in initial rate and equilibrium binding studies that demonstrate pronounced binding antisynergy (21-fold) between PAPS and raloxifene, but not DHEA. Analysis of crystal structures suggests that PAP binding restricts access to the acceptor-binding pocket by restructuring a nine-residue segment of the pocket edge that constricts the active site opening, or "pore", that sieves substrates on the basis of their geometries. In silico docking predicts that raloxifene, which is considerably larger than DHEA, can bind only to the unliganded (open) enzyme, whereas DHEA binds both the open and closed forms. The predictions of these structures with regard to substrate binding are tested using equilibrium and pre-steady-state ligand binding studies, and the results confirm that a nucleotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2A1 and possibly other sulfotransferases.

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.

Cytosolic sulfotransferases (SULTs) are the enzymes that catalyze the sulfonation reaction, an important metabolic pathway for numerous endogenous and exogenous compounds. Human SULTs exhibit complex patterns of broad, differential and overlapping substrate selectivity. Moreover, these enzymes often display substrate inhibition kinetics (i.e., inhibition of the enzyme activity at high substrate concentrations).At present, the crystal structures for 12 human SULTs (i.e., SULT1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, 1E1, 2A1, 2B1a, 2B1b and 4A1) are available, many of which are in complex with a substrate. This review describes the similarities and differences in these structures (particularly the active-site structures) of SULT enzymes. The authors also discuss the structural basis for understanding the catalytic mechanism, the substrate inhibition mechanisms, the cofactor (3'-phosphoadenosine 5'-phosphosulfate or PAPS) binding and the substrate recognition.Correlations of the structural features (including conformational flexibility) in the active sites with the substrate profiles of several SULTs have been well established. One is encouraged to closely integrate in silico approaches with the structural knowledge of the active sites for development of a rationalized and accurate tool that is able to predict metabolism of SULTs toward chemicals and drug candidates.

The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3′-phosphoadenosine-5′-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3′,5′-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.

Sulfation represents an essential modification for various molecules and regulates many biological processes. The sulfation of glycans requires a specific transporter for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) on the Golgi apparatus. This study investigated the expression of PAPS transporter genes in colorectal carcinomas and the significance of Golgi-specific sulfation in the proliferation of colorectal carcinoma cells. The relative amount of PAPST1 transcripts was found to be higher than those of PAPST2 in colorectal cancerous tissues. Immunohistochemically, the enhanced expression of PAPST1 was observed in fibroblasts in the vicinity of invasive cancer cells, whereas the expression of PAPST2 was decreased in the epithelial cells. RNA interference of either of the two PAPS transporter genes reduced the extent of sulfation of cellular proteins and cellular proliferation of DLD-1 human colorectal carcinoma cells. Silencing the PAPS transporter genes reduced fibroblast growth factor signaling in DLD-1 cells. These findings indicate that PAPS transporters play a role in the proliferation of colorectal carcinoma cells themselves and take part in a desmoplastic reaction to support cancer growth by controlling their sulfation status.

Sulfurylation of biomolecules (often termed sulfonation or sulfation) has been described in many organisms in all kingdoms of life. To date, most studies on sulfotransferases, the enzymes catalyzing sulfurylation, have focused on 3'-phosphate-5'-phosphosulfate (PAPS)-dependent enzymes, which transfer the sulfuryl group from this activated anhydride to hydroxyl groups of acceptor molecules. By contrast, the PAPS-independent aryl sulfotransferases (ASSTs) from bacteria, which catalyze sulfotransfer from phenolic sulfate esters to another phenol in the bacterial periplasm, were not well characterized until recently, although they were first described in 1986 in a search for nonhepatic sulfurylation processes. Recent studies revealed that this unusual class of sulfotransferases differs profoundly in both molecular structure and catalytic mechanism from PAPS-dependent sulfotransferases, and that ASSTs from certain bacterial pathogens are upregulated during infection. In this review, we summarize the literature on the roles of sulfurylation in prokaryotes and analyze the occurrence of ASSTs and their dependence on Dsb proteins catalyzing oxidative folding in the periplasm. Furthermore, we discuss structural differences and similarities between aryl sulfotransferases and PAPS-dependent sulfotransferases.

We propose a novel strategy for determining the elemental composition of organic compounds using the peak ratio of isotopic fine structure observed by high-magnetic field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Using 3'-phosphoadenosine 5'-phosphosulfate and CTU guanamine as standard organic compounds, isotopic peaks derived from (15)N-, (34)S-, and (18)O-substituted forms were separated from (13)C-substituted species. Furthermore, these isotopic peaks were quantitatively detected and closely matched the natural abundance of each element. These data successfully led us to determine the one elemental composition in a standard independent manner. The approach should be particularly amenable to the metabolomics research field.

The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1∗3, bound with 3′-phosphoadenosine 5′-phosphate (PAP), at 2.4 and 2.3 A resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1 A further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1∗3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher Km and two times lower Vmax with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.