Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent

A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity.     

Simple Determination of Steroid Sulfatase Activity in Enzyme Preparation

Abstract

Simple one tube method for the determination of steroid sulfatase activity in an enzyme preparation or commercially available preparation is described. Principle of the reaction is as follows; dehydroepi-androsterone sulfate (DHEA-S) as substrate is hydrolyzed by the catalysis of sulfatase and DHEA released is determined by the enzymic method using 3β-hydroxysteroid oxidase.     

Sulfatases: structure, mechanism, biological activity, inhibition, and synthetic utility

Sulfatases, which cleave sulfate esters in biological systems, play a key role in regulating the sulfation states that determine the function of many physiological molecules. Sulfatase substrates range from small cytosolic steroids, such as estrogen sulfate, to complex cell-surface carbohydrates, such as the glycosaminoglycans. The transformation of these molecules has been linked with important cellular functions, including hormone regulation, cellular degradation, and modulation of signaling pathways. Sulfatases have also been implicated in the onset of various pathophysiological conditions, including hormone-dependent cancers, lysosomal storage disorders, developmental abnormalities, and bacterial pathogenesis. These findings have increased interest in sulfatases and in targeting them for therapeutic endeavors. Although numerous sulfatases have been identified, the wide scope of their biological activity is only beginning to emerge. Herein, accounts of the diversity and growing biological relevance of sulfatases are provided along with an overview of the current understanding of sulfatase structure, mechanism, and inhibition.     

Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity

O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [³⁵S] 3′-phosphoadenosine-5′-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.

Hippurathydrolase,ein neues Enzym aus Mikroorganismen, 1. Mitt.: Induzierte Biosynthese in Fusarium semitectum,Darstellung einer gereinigten Enzympräparation und Untersuchungen zur Substratspezifität

WhenFusarium semitectum is grown in media containing hippurate, the synthesis of an enzyme is induced, which effects hydrolytic splitting of hippuric acid to benzoic acid and glycine. A 130-fold purified enzyme preparation is obtained from mycelial extracts by heat treatment, ammonium sulfate fractionation, and adsorption on calcium phosphate gel. Comparative studies of the substrate specificity indicate the presence of a new enzyme. Contrary to aminoacylase (E.C. 3.5.1.14) and related enzymes, the preferred substrates of the new enzyme are hippuric acid and similar N-benzoylated amino acids. The trivial namehippurate hydrolase and the systematic nameN-benzoylaminoacid amidohydrolase is proposed for this enzyme.     

Catalytic Mechanism of the Arylsulfatase Promiscuous Enzyme from Pseudomonas Aeruginosa

To elucidate the working mechanism of the “broad substrate specificity” by the Pseudomonas aeruginosa aryl sulfatase (PAS) enzyme, we present here a full quantum chemical study performed at the density functional level. This enzyme is able to catalyze the hydrolysis of the original p‐nitrophenyl‐sulfate (PNPS) substrate and the promiscuous p‐nitrophenyl‐phosphate (PNPP) one with comparable reaction kinetics. Based on the obtained results, a multistep mechanism including activation of the nucleophile, the nucleophilic attack, and the cleavage of the S? O (P? O) bond is proposed. Regarding the phosphate monoester, the results indicate that only some steps of the promiscuous reaction are identical to those in the native process. Differences concern mainly the last step in which the His115 residue acts as a general base to accept the proton by the O atom of the FGly51 in the PNPS, whereas in PNPP, the Asp317 protonated residue works as a general acid to deliver a proton by a water molecule to the oxygen atom of the C? O bond. The shapes of the relative potential‐ energy surface (PES) are similar in the two examined cases but the rate‐determining step is different (nucleophile attack vs. nucleophile activation). The influence of the dispersion contributions on the investigated reactions was also taken into account.     

A role for sulfation-desulfation in the uptake of bisphenol a into breast tumor cells

Bisphenol A (BPA) is a widely used plasticizer whose estrogenic properties may impact hormone-responsive disorders and fetal development. In vivo, BPA appears to have greater activity than is suggested by its estrogen receptor (ER) binding affinity. This may be a result of BPA sulfation/desulfation providing a pathway for selective uptake into hormone-responsive cells. BPA is a substrate for estrogen sulfotransferase, and bisphenol A sulfate (BPAS) and disulfate are substrates for estrone sulfatase. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Treatment of MCF-7 cells with BPAS leads to desulfation and uptake of BPA. No BPAS is found inside the cells. These findings suggest a mechanism for the selective uptake of BPA into cells expressing estrone sulfatase. Therefore, sulfation may increase the estrogenic potential of xenobiotics.     

A different method of measuring and detecting mono-and dioxygenase activities

A spectrophotometric method of measuring oxygenase activity in cell extracts or in zymograms was developed. It is an easy and cheap method that allows spectrophotometric measurement of activity by a colored reaction and reveals activity bands in a polyacrylamide gel electrophoresis (PAGE) gel as brown bands. To prove its usefulness, we report on a study with the oxygenase present in strain YR-1, isolated from petroleum-contaminated soils, that uses hydrocarbons as its sole carbon source. Soluble oxygenase activity was detected (under our conditions of cellular homogenization) in the mycelium of a filamentous fungus strain named YR-1. Oxygenase activity from aerobically grown mycelium was detected in growth medium containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited similar optimum pH for the hydroxylation of different aliphatic or aromatic substrates (decane, hexadecane, benzene, and naphthalene) to the corresponding alcohols. Zymogram analysis conducted with partially purified fractions from cell extracts from the aerobic mycelium of the YR-1 strain indicated the existence of only one oxygenase enzyme. Partially purified samples of enzyme, analyzed by sodium dodecyl sulfate PAGE, indicated the presence of one major protein band with a mol wt of 56 kDa that can be a constituent of the native enzyme. In samples of the enzyme, the 56-kDa protein gave a positive reaction in immuno-detection experiments with antibodies directed against oxygenase from soybean. The partially purified enzyme oxidized different substrates, although higher activity was displayed with benzene. K _m values obtained for benzene and decane indicated a higher affinity for the latter     

Enzyme–Substrate Binding Landscapes in the Process of Nitrile Biodegradation Mediated by Nitrile Hydratase and Amidase

The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme–substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and l-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme’s binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.     

Sulfated poly-amido-saccharides (sulPASs) are anticoagulants in vitro and in vivo

Anticoagulant therapeutics are a mainstay of modern surgery and of clotting disorder management such as venous thrombosis, yet performance and supply limitations exist for the most widely used agent – heparin. Herein we report the first synthesis, characterization, and performance of sulfated poly-amido-saccharides (sulPASs) as heparin mimetics. sulPASs inhibit the intrinsic pathway of coagulation, specifically FXa and FXIa, as revealed by ex vivo human plasma clotting assays and serine protease inhibition assays. sulPASs activity positively correlates with molecular weight and degree of sulfation. Importantly, sulPASs are not degraded by heparanases and are non-hemolytic. In addition, their activity is reversed by protamine sulfate, unlike small molecule anticoagulants. In an in vivo murine model, sulPASs extend clotting time in a dose dependent manner with bleeding risk comparable to heparin. These findings support continued development of synthetic anticoagulants to address the clinical risks and shortages associated with heparin.

Heparin mimicking sulfated poly-amido-saccharides (sulPASs) are anticoagulants resistant to heparanases and reversed by protamine sulfate. In an in vivo murine model, sulPASs extend clotting time without the increased risk of bleeding.     

The steroid monooxygenase from Rhodococcus rhodochrous; a versatile biocatalyst

The substrate scope of a steroid monooxygenase (STMO) from Rhodococcus rhodochrous DSM 43269 was investigated for a large range of different ketone substrates. These studies revealed that this enzyme not only oxygenates steroids, but also ketone moieties of a series of other open-chain ketones, such as cyclohexyl methyl ketone, cyclopentyl methyl ketone, and 3-acetylindole. Furthermore, the STMO catalyzed the oxygenation of cyclobutanone derivatives. Comparative biotransformations with recombinant Escherichia coli resting cells harboring the STMO, the cycloalkanone monooxygenase (CAMO) from Cylindrocarpon radicicola or the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus revealed that the STMO is enantiodivergent compared to the CHMO-type. Moreover, the STMO resulted in a higher enantiomeric excess of the product lactones compared to the known BVMOs of the same enantiopreference, such as cyclopentanone monooxygenases.     

Contactless conductivity detection for screening myrosinase substrates by capillary electrophoresis

Myrosinase is a unique enzyme that catalyzes the hydrolysis of glucosinolates (GLS) to isothiocyanate (ITC), glucose and sulfate. Isothiocyanates display a diversified very interesting biological activity. In this study, capillary electrophoresis (CE) was used for the first time for evaluating myrosinase kinetics (maximum velocity V_max and Michaelis–Menten constant K_m) and to assess the affinity of a variety of substrates toward this enzyme.     

The use of a thermostable signature amidase in the resolution of the bicyclic synthon (rac)-γ-lactam

The resolution of the bicyclic synthon (rac)-γ-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) is an important step in the synthesis of a group of chemotherapuetic agents known as carbocyclic nucleosides. The archaeon Sulfolobus solfataricus MT4 produces a thermostable γ-lactamase that has a high sequence homology to the signature amidase family of enzymes. It shows similar inhibition patterns of amidases towards benzonitrile, phenylmethylsulfonyl fluoride and heavy metals such as Hg²⁺, and is activated by thiol reagents. The enzyme selectively cleaves the (+)-enantiomer from a racemic mix of γ-lactam. It also exhibits general amidase activity by cleaving linear and branched aliphatic and aromatic amides. The enzyme catalyses the synthesis of benzoic hydrazide from benzamide preferentially to benzamide cleavage in the presence of excess hydrazine. This enzyme has potential for use in industrial biotransformations in the production of both carbocyclic nucleosides and hydrazides.     

Stereospecific capillary electrophoresis assays using pentapeptide substrates for the study of Aspergillus nidulans methionine sulfoxide reductase A and mutant enzymes

Stereospecific capillary electrophoresis‐based methods for the analysis of methionine sulfoxide [Met(O)]‐containing pentapeptides were developed in order to investigate the reduction of Met(O)‐containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N‐acetylated, C‐terminally 2,4‐dinitrophenyl (Dnp)‐labeled pentapeptides ac‐Lys‐Phe‐Met(O)‐Lys‐Lys‐Dnp, ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp and ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp was achieved in 50 mM Tris‐HCl buffers containing sulfated β‐CD in fused‐silica capillaries, while the diastereomer separation of ac‐Lys‐Asp‐Met(O)‐Asp‐Lys‐Dnp was achieved by sulfated β‐CD‐mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild‐type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild‐type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met‐S‐(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.     

Microbial Sulfatases

The capacity for cleaving off modifying sulfated molecules is related to the presence of sulfatase enzymes in all organisms. Sulfatases (EC 3.1.6.) remove sulfate groups from a set of diverse molecules and constitute a quite heterogeneous group of enzymes characterized by diverse catalytic mechanisms. Human sulfatases are characterized in most detail due to their important role in certain physiological processes. Microbial sulfatases remain virtually uncharacterized on the biochemical level and their potential remains unexplored. The present review summarizes the results of research on sulfatases from bacteria and lower eukaryotes, addresses the connection between these results and the established regularities in the current classification of sulfatases, and considers the potential applications for the use of sulfatases.     

An efficient and facile synthesis of inhibitors for hepatitis C viral and anti-SARS agents: 4-aryl-5-cyano-1,6-dihydro-2-thiouracils

One-pot, multicomponent reaction for the synthesis of 4-aryl-5-cyano-1,6-dihydro-2-thiouracils via three-component from aromatic aldehydes, ethyl 2-cyanoacetate and S-benzylisothiourea hydrochloride (methyl carbamimidothioate sulfate) under methanol is described. These compounds have many drug activities, such as anti-hepatitis C viral, anti-Severe acute respiratory syndrome and anti-HIV-1 integrese activity. The advantages of this procedure include the short reaction time, mild reaction conditions and excellent yields.     

Structural Determinants of the Specific Activities of an L-Amino Acid Oxidase from Pseudoalteromonas luteoviolacea CPMOR-1 with Broad Substrate Specificity

The Pseudoalteromonas luteoviolacea strain CPMOR-1 expresses a flavin adenine dinucleotide (FAD)-dependent L-amino acid oxidase (LAAO) with broad substrate specificity. Steady-state kinetic analysis of its reactivity towards the 20 proteinogenic amino acids showed some activity to all except proline. The relative specific activity for amino acid substrates was not correlated only with K_m or k_cat values, since the two parameters often varied independently of each other. Variation in K_m was attributed to the differential binding affinity. Variation in k_cat was attributed to differential positioning of the bound substrate relative to FAD that decreased the reaction rate. A structural model of this LAAO was compared with structures of other FAD-dependent LAAOs that have different substrate specificities: an LAAO from snake venom that prefers aromatic amino acid substrates and a fungal LAAO that is specific for lysine. While the amino acid sequences of these LAAOs are not very similar, their overall structures are comparable. The differential activity towards specific amino acids was correlated with specific residues in the active sites of these LAAOs. Residues in the active site that interact with the amino and carboxyl groups attached to the α-carbon of the substrate amino acid are conserved in all of the LAAOs. Residues that interact with the side chains of the amino acid substrates show variation. This provides insight into the structural determinants of the LAAOs that dictate their different substrate preferences. These results are of interest for harnessing these enzymes for possible applications in biotechnology, such as deracemization.     

Influence of sulfation conditions on the acidic and catalytic properties of sulfated alumina in isobutane alkylation with butylenes and <Emphasis Type="Italic">n</Emphasis>-pentane isomerization

The influence of the sulfation parameters (the source and concentration of sulfate ions) and the calcination temperature on the acidic and catalytic properties of sulfated alumina in the alkylation of isobutane with butylenes and n-pentane isomerization was studied. IR spectroscopy of adsorbed probe molecules and temperature-programmed desorption of ammonia were used to characterize the acidic properties of the catalysts. An increase in the content of sulfate groups to the value corresponding to a formal value of the monolayer capacity increases the activity of alkylation and the concentration of strong Brönsted sites. The dependence of the stability of activity in alkylation on the sulfate group concentration is extreme with a maximum at the concentration close to the monolayer capacity. It was concluded from the IR spectroscopic data that the decrease in the stability of activity with the further increase in the content of sulfate groups is due to an increase in the concentration of strong Lewis sites and/or an increase in the surface density of strong Brönsted sites. The absence of the correlation between the catalytic behavior of sulfated alumina samples in n-pentane isomerization and acidity indicates that paraffin activation on these samples occurs via the non-acidic mechanism.     

Synthesis of Sulfated Polysaccharides and Oligosaccharide Derivatives with Potent Anti-Aids Virus Activity

Abstract

Sulfated synthetic polysaccharides (with both high anti-AIDS virus activity and high anticoagulant activity) were prepared by sulfating such synthetic polysaccharides as ribopyranan, ribofuranans, and dextrans. Sulfated natural polysaccharides with high anti-AIDS virus activity but low anticoagulant activity were synthesized from lentinan and curdlan. It is assumed that curdlan sulfate will be helpful as an AIDS drug. In addition, sulfated alkyl oligosaccharides with high anti-AIDS virus activity were prepared.     

Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2

Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, tyrosylprotein sulfotransferase-1 and-2. These enzymes catalyze the transfer of sulfate from the universal sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residues to form tyrosine O-sulfate ester and PAP. More than 60 proteins have been identified to be tyrosine sulfated including several G protein-coupled receptors, such as CC-chemokine receptor 8 (CCR8) that is implicated in allergic inflammation, asthma, and atherogenesis. However, the kinetic properties of purified tyrosylprotein sulfotransferase (TPST)-1 and −2 have not been previously reported. Moreover, currently there is no available quantitative TPST assay that can directly monitor individual sulfation of a series of tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both isozymes using CCR8 peptides as substrates that have three tyrosine residues in series. Our method can differentiate between mono-and disulfated products, and our results show that the K_m,app for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs and in determining its catalytic mechanism.