Evolution and multiple functions of sulfonation and cytosolic sulfotransferases across species.

Organisms have conversion systems for sulfate ion to take advantage of the chemical features. The use of biologically converted sulfonucleotides varies in an evolutionary manner, with the universal use being that of sulfonate donors. Sulfotransferases have the ability to transfer the sulfonate group of 3'-phosphoadenosine 5'-phosphosulfate to a variety of molecules. Cytosolic sulfotransferases (SULTs) play a role in the metabolism of low-molecular-weight compounds in response to the host organism's living environment. This review will address the diverse functions of the SULT in evolution, including recent findings. In addition to the diversity of vertebrate sulfotransferases, the molecular aspects and recent studies on bacterial and plant sulfotransferases are also addressed.

Construction of a Protein Crystalline Inclusion-Based Enzyme Immobilization System for Biosynthesis of PAPS from ATP and Sulfate.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the bioactive form of sulfate and is involved in all biological sulfation reactions. The enzymatic transformation method for PAPS is promising, but the low efficiency and high cost of enzyme purification and storage restrict its practical applications. Here, we reported PAPS biosynthesis with a protein crystalline inclusion (PCI)-based enzyme immobilization system. First, the in vivo crystalline inclusion protein CipA was identified as an efficient auto-assembly tag for immobilizing the bifunctional PAPS synthase (ASAK). After characterizing the pyrophosphokinase activity of a polyphosphate exonuclease PaPPX from Pseudomonas aeruginosa, and optimizing the linker fragment, auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA were constructed. Then, the auto-assembled enzymes ASAK-PT-CipA and PaPPX-PT-CipA with high stability were co-expressed and immobilized for constructing a transformation system. The highest transformation rate of PAPS from ATP and sulfate reached 90%, and the immobilized enzyme can be reused 10 times. The present work provided a convenient, efficient, and easy to be enlarged auto-immobilization system for PAPS biosynthesis from ATP and sulfate. The immobilization system also represented a new approach to reduce the production cost of PAPS by facilitating the purification, storage, and reuse of related enzymes, and it would boost the studies on biotechnological production of glycosaminoglycans and sulfur-containing natural compounds.

In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

Sulfation of Phenolic Acids: Chemoenzymatic vs. Chemical Synthesis.

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.

Bacterial Aryl Sulfotransferases in Selective and Sustainable Sulfation of Biologically Active Compounds using Novel Sulfate Donors.

Regioselective sulfation of bioactive compounds is a vital and scarcely studied topic in enzyme-catalyzed transformations and metabolomics. The major bottleneck of enzymatic sulfation consists in finding suitable sulfate donors. In this regard, 3'-phosphoadenosine 5'-phosphosulfate (PAPS)-independent aryl sulfotransferases using aromatic sulfate donors are a favored choice due to their cost-effectiveness. This work presents a unique study of five sulfate donors differing in their leaving group pKa values with a new His-tagged construct of aryl sulfotransferase from Desulfitobacterium hafniense (DhAST-tag). DhAST-tag was purified to homogeneity and biochemically characterized. Two new donors (3-nitrophenyl sulfate and 2-nitrophenyl sulfate) were synthesized. The kinetic parameters of these and other commercial sulfates (4-nitrophenyl, 4-methylumbelliferyl, and phenyl) revealed large differences with respect to the structure of the leaving group. These donors were screened for the sulfation of selected flavonoids (myricetin, chrysin) and phenolic acids (gallate, 3,4-dihydroxyphenylacetate). The donor impact on the sulfation regioselectivity and yield was assessed. The obtained regioselectively sulfated compounds are authentic human metabolites required as standards in clinical trials.

From Steroid and Drug Metabolism to Glycobiology, Using Sulfotransferase Structures to Understand and Tailor Function.

Sulfotransferases are ubiquitous enzymes that transfer a sulfo group from the universal cofactor donor 3'-phosphoadenosine 5'-phosphosulfate to a broad range of acceptor substrates. In humans, the cytosolic sulfotransferases are involved in the sulfation of endogenous compounds such as steroids, neurotransmitters, hormones, and bile acids as well as xenobiotics including drugs, toxins, and environmental chemicals. The Golgi associated membrane-bound sulfotransferases are involved in post-translational modification of macromolecules from glycosaminoglycans to proteins. The sulfation of small molecules can have profound biologic effects on the functionality of the acceptor, including activation, deactivation, or enhanced metabolism and elimination. Sulfation of macromolecules has been shown to regulate a number of physiologic and pathophysiological pathways by enhancing binding affinity to regulatory proteins or binding partners. Over the last 25 years, crystal structures of these enzymes have provided a wealth of information on the mechanisms of this process and the specificity of these enzymes. This review will focus on the general commonalities of the sulfotransferases, from enzyme structure to catalytic mechanism as well as providing examples into how structural information is being used to either design drugs that inhibit sulfotransferases or to modify the enzymes to improve drug synthesis. SIGNIFICANCE STATEMENT: This manuscript honors Dr. Masahiko Negishi's contribution to the understanding of sulfotransferase mechanism, specificity, and roles in biology by analyzing the crystal structures that have been solved over the last 25 years.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

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.

Structural basis for the substrate recognition mechanism of ATP-sulfurylase domain of human PAPS synthase 2.

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.

Analysis of 3'-Phosphoadenosine 5'-Phosphosulfate Transporters: Transporter Activity Assay, Real-Time Reverse Transcription Polymerase Chain Reaction, and Immunohistochemistry.

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.

Insights into the substrate binding mechanism of SULT1A1 through molecular dynamics with excited normal modes simulations.

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.

PAP/SAL1 retrograde signaling pathway modulates iron deficiency response in alkaline soils.

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.

Round, round we go - strategies for enzymatic cofactor regeneration.

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.

Identification of Novel α-Pyrones from Conexibacter woesei Serving as Sulfate Shuttles.

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 Conexibacter woesei. Insights into their biosynthesis via a type III polyketide synthase were obtained by feeding studies using isotope-enriched precursors. In vitro assays employing the genetically associated 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase CwoeST revealed conexipyrones as the enzymes' genuine sulfate acceptor substrates. Furthermore, conexipyrones were determined to function as sulfate shuttles in a two-enzyme assay, because their sulfated derivatives were accepted as donor molecules by the PAPS-independent arylsulfate sulfotransferase (ASST) Cpz4 to yield sulfated caprazamycin intermediates.

[Production and application of 3-phosphoadenosine-5- phosphosulfate].

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.

Increased 3'-Phosphoadenosine-5'-phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A.

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.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles.

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.

Environmental Calcium Initiates a Feed-Forward Signaling Circuit That Regulates Biofilm Formation and Rugosity in Vibrio vulnificus.

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 vulnificusV. 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.IMPORTANCE 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.

Sterol Sulfates and Sulfotransferases in Marine Diatoms.

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 (-SO3H) from the universal donor 3'-phosphoadenosine-5'-phosphosulfate to the hydroxyl function at C-3 of the steroid skeleton. The absence of molecular tools has been the main impediment to the development of a biosynthetic study of this class of compounds in marine organisms. In fact, there is very limited information about these enzymes in marine environments. SULT activity has, however, been reported in several marine species, and, recently, the production of sterol sulfates has been linked to the control of growth in marine diatoms. In this chapter, we describe methods for the study of sterol sulfates in this lineage of marine microalgae. The main aim is to provide the tools useful to deal with the biosynthesis and regulation of these compounds and to circumvent the bottleneck of the lack of molecular information. The protocols have been designed for marine diatoms, but most of the procedures can be used for other marine organisms.

Chemoenzymatic synthesis of 3'-phosphoadenosine-5'-phosphosulfate coupling with an ATP regeneration system.

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.

Small Intestinal Absorption of Methylsulfonylmethane (MSM) and Accumulation of the Sulfur Moiety in Selected Tissues of Mice.

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.