Adenosine 5'-phosphosulfate Research Articles

Gut microbiota-mediated C-sulfonate metabolism impairs the bioavailability and anti-cholestatic efficacy of andrographolide

ABSTRACT Cholestatic liver injury results from the accumulation of toxic bile acids in the liver, presenting a therapeutic challenge with no effective treatment available to date. Andrographolide (AP) has exhibited potential as a treatment for cholestatic liver disease. However, its limited oral bioavailability poses a significant obstacle to harnessing its potent therapeutic properties and restricts its clinical utility. This limitation is potentially attributed to the involvement of gut microbiota in AP metabolism. In our study, employing pseudo-germ-free, germ-free and strain colonization animal models, along with 16S rRNA and shotgun metagenomic sequencing analysis, we elucidate the pivotal role played by gut microbiota in the C-sulfonate metabolism of AP, a process profoundly affecting its bioavailability and anti-cholestatic efficacy. Subsequent investigations pinpoint a specific enzyme, adenosine-5’-phosphosulfate (APS) reductase, predominantly produced by Desulfovibrio piger, which catalyzes the reduction of SO4 2- to HSO3 −. HSO3 − subsequently interacts with AP, targeting its C=C unsaturated double bond, resulting in the formation of the C-sulfonate metabolite, 14-deoxy-12(R)-sulfo andrographolide (APM). Inhibition of APS reductase leads to a notable enhancement in AP bioavailability and anti-cholestatic efficacy. Furthermore, employing RNA sequencing analysis and farnesoid X receptor (FXR) knockout mice, our findings suggest that AP may exert its anti-cholestatic effects by activating the FXR pathway to promote bile acid efflux. In summary, our study unveils the significant involvement of gut microbiota in the C-sulfonate metabolism of AP and highlights the potential benefits of inhibiting APS reductase to enhance its therapeutic effects. These discoveries provide valuable insights into enhancing the clinical applicability of AP as a promising treatment for cholestatic liver injury.

#49 Uremic toxins and inflammation: Metabolic pathways affected in stage 5 kidney disease

Abstract Background and Aims Chronic kidney disease (CKD) is a global health threat, affecting around 12% of the population. The uremic phenotype associated with CKD is considered a predictor for the high cardiovascular and overall mortality in these patients and inflammation is regarded as an essential contributor to it. We seek to discover new inflammation-related metabolites in the CKD population through the use of semitargeted metabolomics, in order to gain insight into the pathways involved and identify potential biomarkers. Method The study employed an observational prospective design to investigate metabolite impact on inflammation in 50 non-dialysis-dependent stage 5 CKD patients. Blood samples were collected before the start of the first haemodialysis session. A semitargeted metabolomic approach was followed, utilising liquid chromatography and high-resolution mass spectrometry with an in-house library of polar compounds. Univariate (Mann-Whitney test) and multivariate (logistic regression with LASSO regularization) analyses identified metabolomic variables associated with inflammation. Results Univariate analysis revealed that adenosine-5′-phosphosulfate (APS) (p-value (p) 0.0211), dimethyglycine (p 0.0015), pyruvate (p 0.0066), lactate (p 0.0156) and 2-ketobutyric acid (p 0.0458) were significantly increased with inflammation, with differences over 1,5-fold changes (inflammation = yes vs inflammation = no). Cholic acid (p 0.01655), homogentisic acid (p 0.0007), 2-phenylpropionic acid (p 0.0351) and 3-indoxylsulfate (p 0.0087) show similar patterns but in the opposite direction, as observed in the volcano plot in Fig. 1. Multivariate analysis revealed significant associations between metabolites and inflammation in CKD stage 5 patients. Specifically, odds ratios for N-Butyrylglycine (OR: 1.87), dimethylglycine (OR: 1.81), 2-Oxoisopentanoic acid (OR: 1.70), and pyruvate (OR: 1.71) were related to a higher probability of experiencing inflammation. Conversely, homogentisic acid (OR: 072), 2-Phenylpropionic acid (OR: 0.77), and 2-Methylglutaric acid (OR: 0.78) exhibited an OR<1, suggestive of decreased probability. This is graphically represented in Fig. 2. Conclusion In our semi-targeted metabolomics study on stage 5 CKD patients, we pinpointed various metabolites linked to inflammation. Notably, dimethylglycine, a uremic toxin, may contribute to vascular damage, while pyruvate's abnormal metabolism is a known feature in heart failure and chronic kidney disease. N-butyrilglycine and 2-oxoisopentanoic acid were also associated with inflammation, potentially tied to mitochondrial fatty acid beta oxidation and incomplete breakdown of branched-chain amino acids. APS, impacting bone development and intestinal permeability, showed a positive correlation with inflammation. Conversely, homogentisic acid and phenylpropionic acid were negatively correlated. While homogentisic acid's anti-inflammatory role isn't clear, phenylpropionic acid exhibits protective effects against hepatotoxicity by reducing immune response. 2-methylglutaric acid and cholic acid were inversely correlated with inflammation, suggesting a protective effect related to fatty acid metabolism and bile acid regulation. These identified metabolites enhance our understanding of CKD-associated inflammation and present strong candidates for future biomarker validation with potential for clinical translation.

Uremic Toxins and Inflammation: Metabolic Pathways Affected in Non-Dialysis-Dependent Stage 5 Chronic Kidney Disease.

Chronic kidney disease (CKD) affects approximately 12% of the global population, posing a significant health threat. Inflammation plays a crucial role in the uremic phenotype of non-dialysis-dependent (NDD) stage 5 CKD, contributing to elevated cardiovascular and overall mortality in affected individuals. This study aimed to explore novel metabolic pathways in this population using semi-targeted metabolomics, which allowed us to quantify numerous metabolites with known identities before data acquisition through an in-house polar compound library. In a prospective observational design with 50 patients, blood samples collected before the initial hemodialysis session underwent liquid chromatography and high-resolution mass spectrometer analysis. Univariate (Mann-Whitney test) and multivariate (logistic regression with LASSO regularization) methods identified metabolomic variables associated with inflammation. Notably, adenosine-5'-phosphosulfate (APS), dimethylglycine, pyruvate, lactate, and 2-ketobutyric acid exhibited significant differences in the presence of inflammation. Cholic acid, homogentisic acid, and 2-phenylpropionic acid displayed opposing patterns. Multivariate analysis indicated increased inflammation risk with certain metabolites (N-Butyrylglycine, dimethylglycine, 2-Oxoisopentanoic acid, and pyruvate), while others (homogentisic acid, 2-Phenylpropionic acid, and 2-Methylglutaric acid) suggested decreased probability. These findings unveil potential inflammation-associated biomarkers related to defective mitochondrial fatty acid beta oxidation and branched-chain amino acid breakdown in NDD stage 5 CKD, shedding light on cellular energy production and offering insights for further clinical validation.

Potassium and jasmonic acid —Induced nitrogen and sulfur metabolisms improve resilience against arsenate toxicity in tomato seedlings

Despite the well-known individual roles of potassium (K+) and jasmonic acid (JA) in plant responses to various stresses, their combined effects on improving tolerance to metalloid arsenate (AsV) toxicity and their influence on physiological and biochemical mechanisms remain largely unexplored. Therefore, the present study was conducted to explore the concomitant role of K+ and JA in nitrogen (N) and sulfur (S) metabolisms and regulation of antioxidant system in tomato seedlings under AsV toxicity conditions. The obtained data reveal that K+ and/or JA boosted the tolerance of tomato (Solanum lycopersicum L.) seedlings to AsV toxicity. However, the effect of a combined dose of K+ and JA was found to be more efficient as compared to the alone application. Seedlings subjected to AsV exhibited suppressed morphological attributes (shoot and root length, shoot and root fresh weight, and shoot and root dry weight), and physio-biochemical characteristics. Also, AsV-treated tomato seedlings showed enhanced reactive oxygen species (ROS) and increased the activity of ROS-producing enzymes (O2•— -generating NADPH oxidase activity and H2O2-generating glycolate oxidase] and also a chlorophyll (Chl) degrading enzyme [chlorophyllase (Chlase) activity]. However, the exogenous supply of K+ and JA synergistically inhibited Chl degradation and overproduction of ROS by suppressing their responsible enzymes activity. Moreover, it induced gas exchange parameters and activity of enzymes responsible for the photosynthesis process (carbonic anhydrase and Rubisco), Chl biosynthesis (δ-aminolevulinic acid dehydratase), and suppressed Chlase activity, and overproduction of ROS and their producing enzymes activity. Most interestingly, the combined application of K+ and JA substantially increased N and S content and their assimilation by upregulating the activity of enzymes involved in these key pathways (nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, ATP sulfurylase, adenosine 5-phosphosulfate reductase, O-acetylserine (thiol) lyase and sulfide reductase). The obtained results strongly reveal that the combined application of K+ and JA improved tolerance of tomato seedlings to AsV toxicity by enhancing N and S assimilation pathways and antioxidant system.

Engineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds

Sulfonation as one of the most important modification reactions in nature is essential for many biological macromolecules to function. Development of green sulfonate group donor regeneration systems to efficiently sulfonate compounds of interest is always attractive. Here, we design and engineer two different sulfonate group donor regeneration systems to boost the biosynthesis of sulfated compounds. First, we assemble three modules to construct a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system and demonstrate its applicability for living cells. After discovering adenosine 5’-phosphosulfate (APS) as another active sulfonate group donor, we engineer a more simplified APS regeneration system that couples specific sulfotransferase. Next, we develop a rapid indicating system for characterizing the activity of APS-mediated sulfotransferase to rapidly screen sulfotransferase variants with increased activity towards APS. Eventually, the active sulfonate group equivalent values of the APS regeneration systems towards trehalose and p-coumaric acid reach 3.26 and 4.03, respectively. The present PAPS and APS regeneration systems are environmentally friendly and applicable for scaling up the biomanufacturing of sulfated products.

Deciphering the biodesulfurization pathway employing marine mangrove Bacillus aryabhattai strain NM1-A2 according to whole genome sequencing and transcriptome analyses

In the biogeochemical cycle, sulfur oxidation plays a vital role and is typically referred to as the elemental sulfur or reductive sulfide oxidation process. This study aimed to characterize a subtropical mangrove-isolated bacterial strain using biochemical, whole-genome, and transcriptome sequencing analyses to enhance our understanding of sulfur metabolism and biodegradation from a molecular genetic perspective. Strain NM1-A2 was characterized as Gram-positive and found to have a close molecular phylogenetic relationship with Bacillus aryabhattai. NM1-A2 efficiently converted dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) via a 4S pathway with 95% efficiency, using enzymes encoded by the dsz operon (dszA, dszB, and dszC), which determine monooxygenases (DszA & DszC) and desulfinase (DszB). The whole-genome sequence of NM1-A2 had a length of approximately 5,257,678 bp and included 16 sulfur metabolism-related genes, featuring the ABC transport system, small subunit (ssu) and cysteine (cys) gene families, and adenosine 5′-phosphosulfate (APS) and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis-related genes. Transcriptomic analysis of NM1-A2 using three sulfur groups—magnesium sulfate (MS), sulfur powder (SP), and sodium thiosulfate (ST) resulted in a significant number of differentially expressed genes (1200, 2304, and 2001, respectively). This analysis revealed that intracellular cysteine concentration directly regulated the expression of cys and ssu genes. Sulfate did not directly affect cys gene expression but repressed ssu gene expression. The cys gene expression levels decreased during the conversion of sulfate to sulfide and cysteine. The transcriptomic data was validated by analyzing the expression patterns of NM1-A2 using real-time quantitative PCR validation analysis. The expression levels of cysl, mccB, and nrnA were significantly upregulated, while cysH, metB, and sat were downregulated in the SP, ST, and MS groups, respectively. This research contributes to our understanding of marine mangrove microorganisms' bacterial efficiency through characterization, whole-genome, and transcriptome sequencing-based molecular degradation of organic compounds in the mangrove ecosystem, which may enhance nutrient availability.

Cannabis sativa L. modulates altered metabolic pathways involved in key metabolisms in human breast cancer (MCF-7) cells: A metabolomics study

The present study investigated the ability of Cannabis sativa leaves infusion (CSI) to modulate major metabolisms implicated in cancer cells survival, as well as to induce cell death in human breast cancer (MCF-7) cells. MCF-7 cell lines were treated with CSI for 48 h, doxorubicin served as the standard anticancer drug, while untreated MCF-7 cells served as the control. CSI caused 21.2% inhibition of cell growth at the highest dose. Liquid chromatography–mass spectroscopy (LC-MS) profiling of the control cells revealed the presence of carbohydrate, vitamins, oxidative, lipids, nucleotides, and amino acids metabolites. Treatment with CSI caused a 91% depletion of these metabolites, while concomitantly generating selenomethionine, l-cystine, deoxyadenosine triphosphate, cyclic AMP, selenocystathionine, inosine triphosphate, adenosine phosphosulfate, 5'-methylthioadenosine, uric acid, malonic semialdehyde, 2-methylguanosine, ganglioside GD2 and malonic acid. Metabolomics analysis via pathway enrichment of the metabolites revealed the activation of key metabolic pathways relevant to glucose, lipid, amino acid, vitamin, and nucleotide metabolisms. CSI caused a total inactivation of glucose, vitamin, and nucleotide metabolisms, while inactivating key lipid and amino acid metabolic pathways linked to cancer cell survival. Flow cytometry analysis revealed an induction of apoptosis and necrosis in MCF-7 cells treated with CSI. High-performance liquid chromatography (HPLC) analysis of CSI revealed the presence of cannabidiol, rutin, cinnamic acid, and ferulic. These results portray the antiproliferative potentials of CSI as an alternative therapy for the treatment and management of breast cancer as depicted by its modulation of glucose, lipid, amino acid, vitamin, and nucleotide metabolisms, while concomitantly inducing cell death in MCF-7 cells.

Crystal structure of adenosine 5ʹ-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.

Phosphorylation and sulfation share a common biosynthetic pathway, but extend biochemical and evolutionary diversity of biological macromolecules in distinct ways.

Phosphate and sulfate groups are integral to energy metabolism and introduce negative charges into biological macromolecules. One purpose of such modifications is to elicit precise binding/activation of protein partners. The physico-chemical properties of the two groups, while superficially similar, differ in one important respect—the valency of the central (phosphorus or sulfur) atom. This dictates the distinct properties of their respective esters, di-esters and hence their charges, interactions with metal ions and their solubility. These, in turn, determine the contrasting roles for which each group has evolved in biological systems. Biosynthetic links exist between the two modifications; the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate being formed from adenosine triphosphate (ATP) and adenosine phosphosulfate, while the latter is generated from sulfate anions and ATP. Furthermore, phosphorylation, by a xylosyl kinase (Fam20B, glycosaminoglycan xylosylkinase) of the xylose residue of the tetrasaccharide linker region that connects nascent glycosaminoglycan (GAG) chains to their parent proteoglycans, substantially accelerates their biosynthesis. Following observations that GAG chains can enter the cell nucleus, it is hypothesized that sulfated GAGs could influence events in the nucleus, which would complete a feedback loop uniting the complementary anionic modifications of phosphorylation and sulfation through complex, inter-connected signalling networks and warrants further exploration.

Rhamnolipid increases H2S generation from waste activated sludge anaerobic fermentation: An overlooked concern

Rhamnolipid (RL), one representative biosurfactant, is widely regarded as an economically feasible and environmentally beneficial additive to improve fermentation efficiency and resource recovery from waste activated sludge (WAS). However, its potentially detrimental impact on WAS fermentation such as H2S generation was overlooked previously. This study therefore aims to fill the gap through exploring whether and how the presence of RL affects H2S generation from WAS anaerobic fermentation. Experimental results showed that when RL increased from 0 to 40 mg/g total suspended solids (TSS), the cumulative H2S yield enhanced from 323.6 × 10−4 to 620.3 × 10−4 mg/g volatile suspended solids (VSS). Mechanism analysis showed that RL reduced WAS surface tension, which benefited transformations of organic sulfurs (e.g., aliphatic-S and sulfoxide) and inorganic sulfate from solid to liquid phase. The presence of RL not only reduced the ratio of α-helix/(β-sheet + random coil) and damaged the hydrogen bonding networks of organic sulfurs but also promoted substrate surface charges and cell membrane permeability. These facilitated the contact between hydrolase and organic sulfurs, thereby increasing sulfide production from organic sulfurs hydrolysis. Further investigations showed that RL promoted the expression of key genes (e.g., aprA/B and dsrA/B) involved in the dissimilatory sulfate reduction, which accelerated the reaction of adenosine 5’-phosphosulfate (APS)→ sulfite→ sulfide. Meanwhile, RL inhibited the corresponding key genes such as CysH, and Sir, responsible for assimilatory sulfate reduction (APS→3’-phosphoadenosine-5’phosphosulfate→organosulfur), which reduced substrate competition in favor of H2S production from dissimilatory sulfate reduction. Besides, RL decreased the fermentation pH, which benefited the transformation of HS− to H2S.

Role of aspartic acid residues D87 and D89 in APS kinase domain of human 3′-phosphoadenosine 5′-phosphosulfate synthase 1 and 2b: A commonality with phosphatases/kinases

3′-phosphoadenosine 5′-phosphosulfate (PAPS) is synthesized in two steps by PAPS synthase (PAPSS). PAPSS is comprised of ATP sulfurylase (ATPS) and APS kinase (APSK) domain activities. ATPS combines inorganic sulfate with α-phosphoryl of ATP to form adenosine 5′-phosphosulfate (APS) and PPi. In the second step APS is phosphorylated at 3′-OH using another mole of ATP to form PAPS and ADP catalyzed by APSK. The transfer of gamma-phosphoryl from ATP onto 3′-OH requires Mg2+ and purported to involve residues D87GD89N. We report that mutation of either aspartic residue to alanine completely abolishes APSK activity in PAPS formation. PAPSS is an, unique enzyme that binds to four different nucleotides: ATP and APS on both ATPS and APSK domains and ADP and PAPS exclusively on the APSK domain. The thermodynamic binding and the catalytic interplay must be very tightly controlled to form the end-product PAPS in the forward direction. Though APS binds to ATPS and APSK, in ATPS domain, the APS is a product and for APSK it is a substrate. DGDN motif is absent in ATPS and present in APSK. Mutation of D87 and D89 did not hamper ATPS activity however abolished APSK activity severely. Thus, D87GD89N region is required for stabilization of Mg2+-ATP, in the process of splitting the γ-phosphoryl from ATP and transfer of γ-phosphoryl onto 3′-OH of APS to form PAPS a process that cannot be achieved by ATPS domain. In addition, gamma32P-ATP, trapped phosphoryl enzyme intermediate more with PAPSS2 than with PAPSS1. This suggests inherent active site residues could control novel catalytic differences. Molecular docking studies of hPAPSS1with ATP + Mg2+ and APS of wild type and mutants supports the experimental results.

Microbes mediating the sulfur cycle in the Atlantic Ocean and their link to chemolithoautotrophy.

Only about 10%-30% of the organic matter produced in the epipelagic layers reaches the dark ocean. Under these limiting conditions, reduced inorganic substrates might be used as an energy source to fuel prokaryotic chemoautotrophic and/or mixotrophic activity. The aprA gene encodes the alpha subunit of the adenosine-5'-phosphosulfate (APS) reductase, present in sulfate-reducing (SRP) and sulfur-oxidizing prokaryotes (SOP). The sulfur-oxidizing pathway can be coupled to inorganic carbon fixation via the Calvin-Benson-Bassham cycle. The abundances of aprA and cbbM, encoding RuBisCO form II (the key CO2 fixing enzyme), were determined over the entire water column along a latitudinal transect in the Atlantic from 64°N to 50°S covering six oceanic provinces. The abundance of aprA and cbbM genes significantly increased with depth reaching the highest abundances in meso- and upper bathypelagic layers. The contribution of cells containing these genes also increased from mesotrophic towards oligotrophic provinces, suggesting that under nutrient limiting conditions alternative energy sources are advantageous. However, the aprA/cbbM ratios indicated that only a fraction of the SOP is associated with inorganic carbon fixation. The aprA harbouring prokaryotic community was dominated by Pelagibacterales in surface and mesopelagic waters, while Candidatus Thioglobus, Chromatiales and the Deltaproteobacterium_SCGC dominated the bathypelagic realm. Noticeably, the contribution of the SRP to the prokaryotic community harbouring aprA gene was low, suggesting a major utilization of inorganic sulfur compounds either as an energy source (occasionally coupled with inorganic carbon fixation) or in biosynthesis pathways.

Crystal Structure of the [4Fe-4S] Cluster-Containing Adenosine-5'-phosphosulfate Reductase from Mycobacterium tuberculosis.

Tuberculosis (TB) is the deadliest infectious disease in the world. In Mycobacterium tuberculosis, the first committed step in sulfate assimilation is the reductive cleavage of adenosine-5′-phosphosulfate (APS) to form adenosine-5′-phosphate (AMP) and sulfite by the enzyme APS reductase (APSR). The vital role of APSR in the production of essential reduced-sulfur-containing metabolites and the absence of a homologue enzyme in humans makes APSR a potential target for therapeutic interventions. Here, we present the crystal structure of the [4Fe–4S] cluster-containing APSR from M. tuberculosis (MtbAPSR) and compare it to previously determined structures of sulfonucleotide reductases. We further present MtbAPSR structures with substrate APS and product AMP bound in the active site. Our structures at a 3.1 Å resolution show high structural similarity to other sulfonucleotide reductases and reveal that APS and AMP have similar binding modes. These studies provide structural data for structure-based drug design aimed to combat TB.

3D-QSAR study of adenosine 5’-phosphosulfate (APS) analouges as ligands for APS reductase

Metabolism of sulfur (sulfur assimilation pathway, SAP) is one of the key pathways for the pathogenesis and survival of persistant bacteria, such as Mycobacterium tuberculosis (Mtb), in the latent period. Adenosine 5'-phosphosulfate reductase (APSR) is an important enzyme involved in the SAP, absent from the human body, so it might represent a valid target for development of new antituberculosis drugs. This work aimed to develop 3D-QSAR model based on the crystal structure of APSR from Pseudomonas aeruginosa, which shows high degree of homology with APSR from Mtb, in complex with its substrate, adenosine 5'-phosphosulfate (APS). 3D-QSAR model was built from a set of 16 nucleotide analogues of APS using alignment-independent descriptors derived from molecular interaction fields (MIF). The model improves the understanding of the key characteristics of molecules necessary for the interaction with target, and enables the rational design of novel small molecule inhibitors of Mtb APSR.

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.

Overexpression of ATP sulfurylase improves the sulfur amino acid content, enhances the accumulation of Bowman–Birk protease inhibitor and suppresses the accumulation of the β-subunit of β-conglycinin in soybean seeds

ATP sulfurylase, an enzyme which catalyzes the conversion of sulfate to adenosine 5′-phosphosulfate (APS), plays a significant role in controlling sulfur metabolism in plants. In this study, we have expressed soybean plastid ATP sulfurylase isoform 1 in transgenic soybean without its transit peptide under the control of the 35S CaMV promoter. Subcellular fractionation and immunoblot analysis revealed that ATP sulfurylase isoform 1 was predominantly expressed in the cell cytoplasm. Compared with that of untransformed plants, the ATP sulfurylase activity was about 2.5-fold higher in developing seeds. High-resolution 2-D gel electrophoresis and immunoblot analyses revealed that transgenic soybean seeds overexpressing ATP sulfurylase accumulated very low levels of the β-subunit of β-conglycinin. In contrast, the accumulation of the cysteine-rich Bowman–Birk protease inhibitor was several fold higher in transgenic soybean plants when compared to the non-transgenic wild-type seeds. The overall protein content of the transgenic seeds was lowered by about 3% when compared to the wild-type seeds. Metabolite profiling by LC–MS and GC–MS quantified 124 seed metabolites out of which 84 were present in higher amounts and 40 were present in lower amounts in ATP sulfurylase overexpressing seeds compared to the wild-type seeds. Sulfate, cysteine, and some sulfur-containing secondary metabolites accumulated in higher amounts in ATP sulfurylase transgenic seeds. Additionally, ATP sulfurylase overexpressing seeds contained significantly higher amounts of phospholipids, lysophospholipids, diacylglycerols, sterols, and sulfolipids. Importantly, over expression of ATP sulfurylase resulted in 37–52% and 15–19% increases in the protein-bound cysteine and methionine content of transgenic seeds, respectively. Our results demonstrate that manipulating the expression levels of key sulfur assimilatory enzymes could be exploited to improve the nutritive value of soybean seeds.

Comparative Genomic Analysis Reveals the Metabolism and Evolution of the Thermophilic Archaeal Genus Metallosphaera.

Members of the genus Metallosphaera are widely found in sulfur-rich and metal-laden environments, but their physiological and ecological roles remain poorly understood. Here, we sequenced Metallosphaera tengchongensis Ric-A, a strain isolated from the Tengchong hot spring in Yunnan Province, China, and performed a comparative genome analysis with other Metallosphaera genomes. The genome of M. tengchongensis had an average nucleotide identity (ANI) of approximately 70% to that of Metallosphaera cuprina. Genes sqr, tth, sir, tqo, hdr, tst, soe, and sdo associated with sulfur oxidation, and gene clusters fox and cbs involved in iron oxidation existed in all Metallosphaera genomes. However, the adenosine-5′-phosphosulfate (APS) pathway was only detected in Metallosphaera sedula and Metallosphaera yellowstonensis, and several subunits of fox cluster were lost in M. cuprina. The complete 3-hydroxypropionate/4-hydroxybutyrate cycle and dicarboxylate/4-hydroxybutyrate cycle involved in carbon fixation were found in all Metallosphaera genomes. A large number of gene family gain events occurred in M. yellowstonensis and M. sedula, whereas gene family loss events occurred frequently in M. cuprina. Pervasive strong purifying selection was found acting on the gene families of Metallosphaera, of which transcription-related genes underwent the strongest purifying selection. In contrast, genes related to prophages, transposons, and defense mechanisms were under weaker purifying pressure. Taken together, this study expands knowledge of the genomic traits of Metallosphaera species and sheds light on their evolution.

Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings

Despite numerous reports on the role of nitric oxide (NO) in regulating plants growth and mitigating different environmental stresses, its participation in sulfur (S) -metabolism remains largely unknown. Therefore, we studied the role of NO in S acquisition and S-assimilation in tomato seedlings under low S-stress conditions by supplying NO to the leaves of S-sufficient and S-deficient seedlings. S-starved plants exhibited a substantial decreased in plant growth attributes, photosynthetic pigment chlorophyll (Chl) and other photosynthetic parameters, and activity of enzymes involved in Chl biosynthesis (δ-aminolevulinic acid dehydratase), and photosynthetic processes (carbonic anhydrase and RuBisco). Also, S-deficiency enhanced reactive oxygen species (ROS) (superoxide and hydrogen peroxide) and lipid peroxidation (malondialdehyde) levels in tomato seedlings. Contrarily, foliar supplementation of NO to S-deficient seedlings resulted in considerably reduced ROS formation in leaves and roots, which alleviated low S-stress-induced lipid peroxidation. However, exogenous NO enhanced proline accumulation by increasing proline metabolizing enzyme (Δ1-pyrroline-5-carboxylate synthetase) activity and also increased NO, hydrogen sulfide (a gasotransmitter small signaling molecule) and S uptake, and content of S-containing compounds (cysteine and reduced glutathione). Under S-limited conditions, NO improved S utilization efficiency of plants by upregulating the activity of S-assimilating enzymes (ATP sulfurylase, adenosine 5-phosphosulfate reductase, sulfide reductase and O-acetylserine (thiol) lyase). Under S-deprived conditions, improved S-assimilation of seedlings receiving NO resulted in improved redox homeostasis and ascorbate content through increased NO and S uptake. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger) invalidated the effect of NO and again caused low S-stress-induced oxidative damage, confirming the beneficial role of NO in seedlings under S-deprived conditions. Thus, exogenous NO enhanced the tolerance of tomato seedlings to limit S-triggered oxidative stress and improved photosynthetic performance and S assimilation.

C-terminal Redox Domain of Arabidopsis APR1 is a Non-Canonical Thioredoxin Domain with Glutaredoxin Function.

Sulfur is an essential nutrient that can be converted into utilizable metabolic forms to produce sulfur-containing metabolites in plant. Adenosine 5′-phosphosulfate (APS) reductase (APR) plays a vital role in catalyzing the reduction of activated sulfate to sulfite, which requires glutathione. Previous studies have shown that the C-terminal domain of APR acts as a glutathione-dependent reductase. The crystal structure of the C-terminal redox domain of Arabidopsis APR1 (AtAPR1) shows a conserved α/β thioredoxin fold, but not a glutaredoxin fold. Further biochemical studies of the redox domain from AtAPR1 provided evidence to support the structural observation. Collectively, our results provide structural and biochemical information to explain how the thioredoxin fold exerts the glutaredoxin function in APR.

Characterization of Entamoeba histolytica adenosine 5'-phosphosulfate (APS) kinase; validation as a target and provision of leads for the development of new drugs against amoebiasis.

BackgroundAmoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed.Methodology/Principal findingsHere, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5′-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica.Conclusions/SignificanceHence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules.