Phosphate Analogues Research Articles

Purification, sequencing and characterization of phospholipase D from Indian mustard seeds

Phospholipase D (PLD; E.C. 3.1.4.4) is widespread in plants where it fulfills diverse functions in growth and in the response to stresses. The enzyme occurs in multiple forms that differ in their biochemical properties. In the present paper PLD from medicinally relevant Indian mustard seeds was purified by Ca2+-mediated hydrophobic interaction and anion exchange chromatography to electrophoretic homogeneity. Based on mass-spectrometric sequence analysis of tryptic protein fragments, oligonucleotide primers for cloning genomic DNA fragments that encoded the enzyme were designed and used to derive the complete amino acid sequence of this PLD. The sequence data, as well as the molecular properties (molecular mass of 92.0kDa, pI 5.39, maximum activity at pH 5.5–6.0 and Ca2+ ion concentrations ⩾60mM), allowed the assignment of this enzyme to the class of α-type PLDs. The apparent kinetic parameters Vmax and Km, determined for the hydrolysis of phosphatidylcholine (PC) in an aqueous mixed-micellar system were 356±15μmolmin−1mg−1 and 1.84±0.17mM, respectively. Phosphate analogs such as NaAlF4 and Na3VO4 displayed strong inhibition of the enzyme. Phosphatidylinositol 4,5-bisphosphate had a strong activating effect at 2–10mM CaCl2. PLD was inactivated at temperatures >45°C. The enzyme exhibited the highest activity toward PC followed by phosphatidylethanolamine and phosphatidylglycerol. PCs with short-chain fatty acids were better substrates than PCs with long fatty acid chains. Lyso-PC was not accepted as substrate.

Gene transcription, metabolite and lipid profiling in eco-indicator daphnia magna indicate diverse mechanisms of toxicity by legacy and emerging flame-retardants.

The use of chemical flame-retardants (FR) in consumer products has steadily increased over the last 30 years. Toxicity data exist for legacy FRs such as pentabromodiphenyl ether (pentaBDE), but less is known about effects of new formulations. To address this issue, the toxicity of seven FR chemicals and formulations was assessed on the freshwater crustacean Daphnia magna. Acute 48-h nominal LC50 values for penta- and octabromodiphenyl ether (pentaBDE, octaBDE), Firemaster 550 (FM550), Firemaster BZ-54 (BZ54), bis(2-ethylhexyl) tetrabromophthalate (BEH-TEBP), triphenyl phosphate (TPhP), and nonbrominated BEH-TEBP analog bis(2-ethylhexyl) phthalate (BEHP) ranged from 0.058 mg/L (pentaBDE) to 3.96 mg/L (octaBDE). mRNA expression, (1)H NMR-based metabolomic and lipidomic profiling at 1/10 LC50 revealed distinct patterns of molecular response for each exposure, suggesting pentaPBDE affects transcription and translation, octaBDE and BEH-TEBP affect glycosphingolipid biosynthesis and BZ54 affects Wnt and Hedgehog signal pathways as well as glycosaminoglycan degradation. Brominated components of FM550 (i.e., BZ54) were significantly higher in Daphnia after 48 h following 1/10 LC50 exposure. FM550 elicited significant mRNA changes at five concentrations across a range from 1/10(6) LC50 to 1/2 LC50. Analyses suggest FM550 impairs nutrient utilization or uptake in Daphnia.

A member of the Phosphate transporter 1 (Pht1) family from the arsenic-hyperaccumulating fern Pteris vittata is a high-affinity arsenate transporter.

Pteris vittata exhibits enhanced arsenic uptake, but the corresponding mechanisms are not well known. The prevalent form of arsenic in most soils is arsenate, which is a phosphate analog and a substrate for Phosphate transporter 1 (Pht1) transporters. Herein we identify and characterize three P. vittata Pht1 transporters. Pteris vittata Pht1 cDNAs were isolated and characterized via heterologous expression in Saccharomyces cerevisiae (yeast) and Nicotiana benthamiana leaves. Expression of the PvPht1 loci in P. vittata gametophytes was also examined in response to phosphate deficiency and arsenate exposure. Expression of each of the PvPht1 cDNAs complemented the phosphate uptake defect of a yeast mutant. Compared with yeast cells expressing Arabidopsis thaliana Pht1;5, cells expressing PvPht1;3 were more sensitive to arsenate, and accumulated more arsenic. Uptake assays with yeast cells and radiolabeled (32)P revealed that PvPht1;3 and AtPht1;5 have similar affinities for phosphate, but the affinity of PvPht1;3 for arsenate is much greater. In P. vittata gametophytes, PvPht1;3 transcript levels increased in response to phosphate (Pi) deficiency and arsenate exposure. PvPht1;3 is induced by Pi deficiency and arsenate, and encodes a phosphate transporter that has a high affinity for arsenate. PvPht1;3 probably contributes to the enhanced arsenate uptake capacity and affinity exhibited by P. vittata.

How release of phosphate from mammalian F1-ATPase generates a rotary substep

The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the α(E)β(E)-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the β(E)-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the β(E)-subunit disrupts interactions in the α(E)β(E-)catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the α(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°.

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca²⁺ Release.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20-500-fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca(2+) with an EC50 of 31 μM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced.

Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity.

The ss-siRNA activity in vivo requires a metabolically stable 5′-phosphate analog. In this report we used crystal structure of the 5′-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5′-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5′-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.

Iron and phosphorus speciation in Fe-conditioned membrane bioreactor activated sludge

Iron dosing of membrane bioreactors (MBRs) is widely used as a means of meeting effluent phosphorus targets but there is limited understanding of the nature of iron and phosphorus-containing solids that are formed within the bioreactor (an important issue in view of the increasing interest in recovering phosphorus from wastewaters). Of particular challenge is the complexity of the MBR system and the variety of reactions that can occur on addition of iron salts to a membrane bioreactor. In this study, the performances of bench scale MBRs with dosing of either ferrous or ferric salts were monitored for a period of four months. The distributions of Fe and P-species in the Fe-conditioned sludges were determined using X-ray absorption spectroscopy (XAS) at the Fe K-edge and the P K-edge. Regardless of whether iron was dosed to the anoxic or aerobic chambers and regardless of whether ferrous (Fe(II)) or ferric (Fe(III)) iron was dosed, iron present in the minerals in the conditioned sludges was consistently in the +III oxidation state. Fitting of the Fe K-edge EXAFS spectra revealed that an Fe(III)-phosphate species was the main Fe species present in all cases with the remaining fraction dominated by lepidocrocite (γ-FeOOH) in the Fe(II)-dosed case and ferrihydrite (am-FeOOH) in the Fe(III)-dosed case. Approximately half the phosphorus in the activated sludge samples was present as a distinct Fe-PO4 mineral (such as strengite or an amorphous ferric hydroxyl phosphate analogue of strengite) and half as phosphorus adsorbed to an iron oxyhydroxide mineral phase indicating that both co-precipitation and adsorption of phosphorus by iron contribute to removal of phosphorus from the MBR supernatant.

Synthesis of hydrolysis-resistant pyridoxal 5′-phosphate analogs and their biochemical and X-ray crystallographic characterization with the pyridoxal phosphatase chronophin

A set of phosphonic acid derivatives (1–4) of pyridoxal 5′-phosphate (PLP) was synthesized and characterized biochemically using purified murine pyridoxal phosphatase (PDXP), also known as chronophin. The most promising compound 1 displayed primarily competitive PDXP inhibitory activity with an IC50 value of 79μM, which was in the range of the Km of the physiological substrate PLP. We also report the X-ray crystal structure of PDXP bound to compound 3, which we solved to 2.75Å resolution (PDB code 5AES). The co-crystal structure proves that compound 3 binds in the same orientation as PLP, and confirms the mode of inhibition to be competitive. Thus, we identify compound 1 as a PDXP phosphatase inhibitor. Our results suggest a strategy to design new, potent and selective PDXP inhibitors, which may be useful to increase the sensitivity of tumor cells to treatment with cytotoxic agents.

Phosphate and thiophosphate biphenyl analogs as steroid sulfatase inhibitors.

In the present work, we report convenient methods for the synthesis and biological evaluation of phosphate and thiophosphate biphenyl derivatives exhibiting steroid sulfatase (STS) activity. The described synthesis is based on straightforward preparation of biphenyl-4-ol and 4'-hydroxy-biphenyl-4-carboxylic acid ethyl ester modified with various phosphate or thiophosphate moieties. The inhibitory effects of these compounds were tested on STS isolated from human placenta and led to two compounds of interest, 5a and 5d with IC50 values of 28.0 and 22.1 µM, respectively and that had interesting new binding modes in the STS active site.

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.

Rat hormone sensitive lipase inhibition by cyclipostins and their analogs

Cyclipostins are bicyclic lipophilic phosphate natural products. We report here that synthesized individual diastereomers of cyclipostins P and R have nanomolar IC50s toward hormone sensitive lipase (HSL). The less potent diastereomers of these compounds have 10-fold weaker IC50s. The monocyclic phosphate analog of cyclipostin P is nearly as potent as the bicyclic natural product. Bicyclic phosphonate analogs of both cyclipostins exhibit IC50s similar to those of the weaker diastereomer phosphates (about 400nM). The monocyclic phosphonate analog of cyclipostin P has similar potency. A series of monocyclic phosphonate analogs in which a hydrophobic tail extends from the lactone side of the ring are considerably poorer inhibitors, with IC50s around 50μM. Finally cyclophostin, a related natural product inhibitor of acetylcholinesterase (AChE) that lacks the hydrocarbon tail of cyclipostins, is not active against HSL. These results indicate a critical SAR for these compounds, the hydrophobic tail. The smaller lactone ring is not critical to activity, a similarity shared with cyclophostin and AChE. The HSL kinetics of inhibition for the cyclipostin P trans diastereomer were examined in detail. The reaction is irreversible with a KI of 40nM and a rate constant for inactivation of 0.2min−1. These results are similar to those observed for cyclophostin and AChE.

Formation of the Motor Protein-Photochromic ADP Analogue-Fluorometal Ternary Complex and Photo-Reversible Transition along the Steps in ATPase Cycle

In the presence of Mg2+ADP, myosin forms stable ternary complexes with phosphate analogues, fluoroberyllate (BeFn), fluoroaluminate (AlF4-) and orthovanadate (Vi), each of which may mimic different transient state along ATPase cycle. It is known that kiensin also forms similar ternary complexes. Our previous studies on the photoaffinity-labelling using photoactive ADP analogue and 19F NMR spectroscopy using 19F-labeled ADP analogue for the ternary complexes revealed that there is some variation in the myosin-nucleotide contacts at the nucleotide base among the ternary complexes. Previously, we have incorporated photochromic molecule, azobenzen derivative into the functional region of ATP driven motor proteins and succeeded to control activities and conformation reversibly by photochromic irradiation. Aim of this study is to induce transition between the different steps in ATPase cycle of motor proteins utilizing photoisomerization on the motor protein-phtochromic ADP analogue-fluorometal ternary complexes. In this study, three types of photochromic ATP analogues composed of azobenznene, spiropyran and fulgimide derivatives were synthesized. The photo-responsive interaction of the photochromic ATP analogues with myosin and kinesin were examined. Photo-dependent conformational changes of the myosin motor domain were monitored by X-ray small angle scattering. And the photo-reversible changes in the velocity of the microtubule glyding for kinesin in the presence of the photochromic ATP analogues were also observed. In the presence of fluorometal, BeFn, photochromic ADP analogues were trapped within the ATPase site of kinesin and myosin resulted in formation of ternary complexes. The conformational changes of the complexes induced by photo irradiation were studied.

First synthesis of diethyl N-acetyl-glycosamine-1- difluoromethylphosphonate from 2-nitroglycals as phosphate analog

First synthesis of diethyl N-acetyl-glycosamine-1- difluoromethylphosphonate from 2-nitroglycals as phosphate analog

Synthesis and steroid sulfatase inhibitory activities of N-alkanoyl tyramine phosphates and thiophosphates

A series of phosphate and thiophosphate analogs based on the frameworks of N-alkanoyl tyramines have been synthesized and biologically evaluated.

Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay.

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

Na+/K+ Pump Ion Binding Site Interactions Regulate the Proton Leak Pathway

The Na+/K+ pump (NKA) is a membrane bound transporter located in animal cells which transports three Na+ ions out for two K+ ions into the cell for every one ATP molecule hydrolyzed. Two out-of-three ion-binding sites within the protein reciprocally bind two K+ or two Na+ ions (shared sites), while the remaining site exclusively binds Na+. Without these two ions present the NKA passively transports H+, likely through the Na-exclusive site. In order to understand the proton transport mechanism voltage-clamp was used to study the effect of tetrapropylammonium (TPA) and Beryllium Fluoride (BeFx) on inward proton current (IH) transport by NKA expressed in Xenopus oocytes. TPA (a shared site blocker) had a dual effect on IH; between pH 7.6-6.0 TPA partially inhibited IH, and at pH 5.0 (where an inhibitory effect on the steady state IH is present), TPA partially activated IH. When the phosphate analogue BeFx was injected into oocytes, stabilizing an externally open conformation of the ion binding sites, K+ still inhibited inward current at pH 7.6. At pH 5.0 K+ activated IH resembling the effect of TPA on fully functional NKA. These results illustrate the complex interaction between the occupancy of shared sites and proton permeation. Blocking access at the shared sites with TPA activated IH by relieving the inhibition normally observed at high [H+]. Partial inhibition by TPA at pH 5.0 may reflect incomplete block of H+ access or a reduced rate of the conformational changes needed for H+ translocation. After BeFx inhibition NKA was still capable of interacting with K+. The mixed occupancy of K+ and H+ at low pH activated the leak, likely relieving the H+ inhibition due to K+ occlusion. Supported by NIH GM 061583 to CG and NSF MCB-1243842 to PA.

Synthesis of Highly Water-Soluble Adamantyl Phosphoinositide Derivatives

Phosphatidylinositol phosphates are key regulators of cell signalling pathways and membrane trafficking in eukaryotic cells, and there is a need for new chemical probes to further understand how they interact with lipid-binding proteins. Here, the synthesis of phosphatidylinositol phosphate analogues containing adamantyl carboxylic ester groups, in place of the natural lipid side chains, is described. These derivatives are considerably more soluble in water than analogues containing other lipid side chains and do not form large aggregates such as liposomes or micelles. These adamantyl analogues bind to known phosphoinositide-binding proteins with similar affinities to native ligands and will facilitate future studies on the substrate specificities of these proteins involving cocrystallisation studies with proteins.

Genome-wide association mapping identifies a new arsenate reductase enzyme critical for limiting arsenic accumulation in plants.

Author SummaryArsenic is a human carcinogen that accumulates from soil into many different food crops, where it presents a significantly increased cancer risk when foods derived from these crops are consumed. Plants naturally control the amount of arsenic they accumulate by first chemically converting arsenate into arsenite, which is then extruded from the roots back into the soil. Because arsenate is a chemical analogue of phosphate, conversion of arsenate in the root to arsenite may also prevent arsenic being efficiently transported to the shoots via the phosphate transport system. The chemical reduction of arsenate to generate arsenite is therefore clearly a key component of a plant's detoxification strategy. Here, we use genetic methods to identify the enzyme responsible for this crucial reaction—HAC1. We show that HAC1 is responsible for arsenate reductase activity in both the outer layer of the root (epidermis) and the inner layer adjacent to the xylem (pericycle). In its absence, the roots return less arsenic to the soil and the shoots accumulate up to 300 times more arsenic. This knowledge creates new opportunities to limit arsenic accumulation in food crops, thereby helping to reduce the cancer risk from this food-chain contaminant.

ChemInform Abstract: Synthesis of Nucleoside Phosphate and Phosphonate Prodrugs

AbstractReview: 390 refs.

First synthesis of diethyl N-acetyl-glycosamine-1-difluoromethylphosphonate from 2-nitroglycals as phosphate analog

Herein, we report the first access to diethyl glycosamine-1-difluoromethylphosphate as phosphate mimic. The diethyl 2-nitro-glycosides-β-1-difluoromethylphosphonate were obtained from a Michael addition of diethyl(difluoro-(trimethylsilyl)methyl)phosphonate on 2-nitroglycals in the presence of a fluoride promoter in good to excellent yields. A subsequent reduction of the nitro group leads to the N-acetyl glycosides derivative giving a straightforward access to phosphate analogs.