Sulfatase Activity Research Articles

Identification and evolution of glucosinolate sulfatases in a specialist flea beetle

Glucosinolates, a characteristic group of specialized metabolites found in Brassicales plants, are converted to toxic isothiocyanates upon herbivory. Several insect herbivores, including the cabbage stem flea beetle (Psylliodes chrysocephala), prevent glucosinolate activation by forming desulfo-glucosinolates. Here we investigated the molecular basis of glucosinolate desulfation in P. chrysocephala, an important pest of oilseed rape. Enzyme activity assays with crude beetle protein extracts revealed that glucosinolate sulfatase (GSS) activity is associated with the gut membrane and has narrow substrate specificity towards the benzenic glucosinolate sinalbin. In agreement with GSS activity localization in vivo, we identified six genes encoding arylsulfatase-like enzymes with a predicted C-terminal transmembrane domain, of which five showed GSS activity upon heterologous expression in insect cells. PcGSS1 and PcGSS2 used sinalbin and indol-3-ylmethyl glucosinolate as substrates, respectively, whereas PcGSS3, PcGSS4, and PcGSS5 showed weak activity in enzyme assays. RNAi-mediated knock-down of PcGSS1 and PcGSS2 expression in adult beetles confirmed their function in vivo. In a phylogenetic analysis of coleopteran and lepidopteran arylsulfatases, the P. chrysocephala GSSs formed a cluster within a coleopteran-specific sulfatase clade distant from the previously identified GSSs of the diamondback moth, Plutella xylostella, suggesting an independent evolution of GSS activity in ermine moths and flea beetles.

Características de la actividad enzimática y el humus en suelos de chinampa.

The chinampas are protected natural areas that have suffered degradation due to saline contamination and type of management. The objective was to study enzymatic activity and type of humus in chinampas with different salinity, land use and geographical location. Forty-five soil samples were studied, 15 at each site (Xochimilco, San Gregorio Atlapulco (SGA) and Tláhuac). Each site had a different use: five had pastures, five had agricultural crops and five were abandoned. pH, EC, saline ions, enzymatic activity (glucosidase, phosphatase, urease, sulfatase, catalase and dehydrogenase) and carbon in humic substances were evaluated. EC, PSI and pH classified the cultivated soils of SGA and Xochimilco as saline, but in Tláhuac the soils of abandoned and pasture chinampas were sodic saline. Geographic location and land use changed enzyme activity (except for urease and glucosidase) and characteristics of the humus [percentages of carbon of humic acids (HA) (humic acids), fulvic acids (FA), and humins]. There were statistical differences (Tukey P < 0.05). The cultivated soils had more urease, phosphatase and sulfatase activity, while in the pasturelands dehydrogenase and catalase were more active. Higher carbon content in AH was found in pastureland and chinampas of Tlahuac, while in SGA and abandoned chinampas carbon content was higher in FA.

Source of organic detritus and bivalve biomass influences nitrogen cycling and extracellular enzyme activity in estuary sediments

In aquatic ecosystems, natural processes that remove nitrogen from the biologically available pool (e.g. denitrification) have been intensively studied as an ecosystem function that reduces eutrophication. The quantity of sediment organic matter is a key driver of denitrification with percent organic content positively related to rates of nitrogen removal; however, few studies have investigated the influence of the quality of organic matter on nitrogen cycling in estuarine sediments despite shifts in primary producers with eutrophication. This laboratory study using intact benthic communities investigates the influence of various organic detritus sources, which vary in their C:N ratio, on nitrogen gas (N2) and solute fluxes and extracellular enzyme activity in estuarine sediments. A custom-built tank with a removable front plate was used with a planar optode film to image sediment oxygenation. Mangrove leaf detritus significantly increased the net N2 production in sediments, while the deposition of other detrital sources and control sediments produced net N2 consumption. Sulfatase activity was significantly reduced in the mangrove leaves and seagrass treatments, suggesting alteration of heterotrophic microbial activity with reducing oxygen conditions. Leucine aminopeptidase activity, indicating nitrogen cycling, was reduced in all treatments, suggesting the organic detritus provided a nitrogen supplement or reduced the activity of extracellular enzymes producing microbes. Bivalve biomass increased net nitrogen gas fluxes in some treatments. Our results indicate different detrital sources may have varying impacts on the removal of bioavailable nitrogen through denitrification and show that feedbacks in biogeochemical cycles may occur with changes in organic detrital source pools.

Heparan Sulfate-Editing Extracellular Sulfatases Enhance VEGF Bioavailability for Ischemic Heart Repair.

Mechanistic insight into the inflammatory response after acute myocardial infarction may inform new molecularly targeted treatment strategies to prevent chronic heart failure. We identified the sulfatase SULF2 in an in silico secretome analysis in bone marrow cells from patients with acute myocardial infarction and detected increased sulfatase activity in myocardial autopsy samples. SULF2 (Sulf2 in mice) and its isoform SULF1 (Sulf1) act as endosulfatases removing 6-O-sulfate groups from heparan sulfate (HS) in the extracellular space, thus eliminating docking sites for HS-binding proteins. We hypothesized that the Sulfs have a role in tissue repair after myocardial infarction. Both Sulfs were dynamically upregulated after coronary artery ligation in mice, attaining peak expression and activity levels during the first week after injury. Sulf2 was expressed by monocytes and macrophages, Sulf1 by endothelial cells and fibroblasts. Infarct border zone capillarization was impaired, scar size increased, and cardiac dysfunction more pronounced in mice with a genetic deletion of either Sulf1 or Sulf2. Studies in bone marrow-chimeric Sulf-deficient mice and Sulf-deficient cardiac endothelial cells established that inflammatory cell-derived Sulf2 and endothelial cell-autonomous Sulf1 promote angiogenesis. Mechanistically, both Sulfs reduced HS sulfation in the infarcted myocardium, thereby diminishing Vegfa (vascular endothelial growth factor A) interaction with HS. Along this line, both Sulfs rendered infarcted mouse heart explants responsive to the angiogenic effects of HS-binding Vegfa164 but did not modulate the angiogenic effects of non-HS-binding Vegfa120. Treating wild-type mice systemically with the small molecule HS-antagonist surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide, 1 mg/kg/day) for 7 days after myocardial infarction released Vegfa from HS, enhanced infarct border-zone capillarization, and exerted sustained beneficial effects on cardiac function and survival. These findings establish HS-editing Sulfs as critical inducers of postinfarction angiogenesis and identify HS sulfation as a therapeutic target for ischemic tissue repair.

Melatonin Enhances the Usefulness of Ionizing Radiation: Involving the Regulation of Different Steps of the Angiogenic Process.

Radiotherapy is a part of cancer treatment. To improve its efficacy has been combined with radiosensitizers such as antiangiogenic agents. Among the mechanisms of the antitumor action of melatonin are antiangiogenic effects. Our goal was to investigate whether melatonin may modulate the sensitivity of endothelial cells (HUVECs) to ionizing radiation. Melatonin (1 mM) enhanced the inhibition induced by radiation on different steps of the angiogenic process, cell proliferation, migration, and tubular network formation. In relation with the activity and expression of enzymes implicated in estrogen synthesis, in co-cultures HUVECs/MCF-7, radiation down-regulated aromatase mRNA expression, aromatase endothelial-specific promoter I.7, sulfatase activity and expression and 17β-HSD1 activity and expression and melatonin enhanced these effects. Radiation and melatonin induced a significant decrease in VEGF, ANG-1, and ANG-2 mRNA expression. In ANG-2 and VEGF mRNA expression melatonin potentiated the inhibitory effect induced by radiation. In addition, melatonin counteracted the stimulatory effect of radiation on FGFR3, TGFα, JAG1, IGF-1, and KDR mRNA expression and reduced ANPEP expression. In relation with extracellular matrix molecules, radiation increased MMP14 mRNA expression and melatonin counteracted the stimulatory effect of radiation on MMP14 mRNA expression and increased TIMP1 expression, an angiogenesis inhibitor. Melatonin also counteracted the stimulatory effect of radiation on CXCL6, CCL2, ERK1, ERK2, and AKT1 mRNA expression and increased the inhibitory effect of radiation on NOS3 expression. In CAM assay, melatonin enhanced the reduction of the vascular area induced by radiation. Melatonin potentiated the inhibitory effect on the activation of p-AKT and p-ERK exerted by radiation. Antiangiogenic effect of melatonin could be mediated through AKT and ERK pathways, proteins involved in vascular endothelial (VE) cell growth, cell proliferation, survival, migration, and angiogenesis. In addition, radiation increased endothelial cell permeability and melatonin counteracted it by regulating the internalization of VE-cadherin. Radiation has some side effects on angiogenesis that may reduce its effectiveness against tumor growth and melatonin is able to neutralize these negative actions of radiation. Additionally, melatonin potentiated radiation-induced antiangiogenic actions on several steps of the angiogenic process and enhanced its antitumor action. Our findings point to melatonin as a useful molecule as adjuvant to radiotherapy in cancer treatment.

Changes of soil enzyme activities and nutrients across different succession stages of grazing alpine Kobresia grassland.

The variation of soil enzyme activity and relevance with soil nutrients was examined in multistable grazing alpine Kobresia grassland, including Gramineae-Kobresia humilis community, K. humilis community, K. pygmaea community at thickened stage, K. pygmaea community at cracked stage and forb-black soil type secondary bare land. The results showed that the vegetation coverage and aboveground biomass successively decreased with degenerative succession. The belowground biomass was the highest in the K. pygmaea community at thickened and cracked stages. The activities of soil sucrase, urease, cellulase, alkaline phosphatase and aryl sulfatase were higher at the surface soil layer (0-10 cm) than those at the subsurface soil layer (10-20 cm), while the pattern of chitinase activity was contrary. The activities of cellulase, alkaline phosphatase and aryl sulfatase were the highest in the Gramineae-K. humilis community and the lowest at the forb-black soil type secondary bare land, and they slightly increased during the thickened stage of K. pygmaea community. Chitinase activity was relatively high at the middle three stages, while urease and sucrase activity had an obvious increase in the forb-black soil type secondary bare land. Soil moisture, ammonium, alkali-hydrolyzable nitrogen, total nitrogen, total carbon and organic carbon successively decreased with degenerative succession, whereas the concentrations of nitrate and available phosphorus increased at the latter two succession stages. The activities of the other enzymes, except for chitinase, were significantly positively correlated with the soil available phosphorus, ammonium, alkali-hydrolyzed nitrogen, total carbon, and organic carbon, and negatively correlated with soil pH. The activities of cellulose, alkaline phosphatase and aryl sulfatase were significantly positively correlated with soil moisture and total nitrogen. The main factors affecting soil enzyme activity were available phosphorus and ammonium. Soil enzymes showed different evolutionary trends influenced by grazing degradation succession in the alpine grassland, with a synergistic effect with soil nut-rients. Moreover, severely degraded extreme environments may stimulate soil enzyme activities related to nitrogen and carbon transformation.

Measurement of recombinant human arylsulfatase A and leukocyte sulfatase activities by analytical isotachophoresis

Metachromatic Leukodystrophy (MLD) and Multiple Sulfatase Deficiency (MSD) are rare and ultra-rare lysosomal storage diseases. Due to enzyme defects, patients are unable to split the sulfategroup from the respective substrates. In MSD all sulfatases are affected due to a defect of the Sulfatase Modifying Factor 1 (SUMF1) gene coding for the formylglycine generating enzyme (FGE) necessary for the modification of the active site of sulfatases. In MLD mutations in the arylsulfatase A (ARSA) gene cause ARSA deficiency with subsequent accumulation of 3-sulfogalactocerebroside especially in oligodendrocytes. The clinical consequence is demyelination and a devastating neurological disease. Enzyme replacement therapy (ERT) with recombinant human arylsulfatase A (rhARSA), gene therapy, and stem cell transplantation are suggested as new therapeutic options. The aim of our study was to characterize rhARSA concerning its substrate specificity using analytical isotachophoresis (ITP). Substrate specificity could be demonstrated by sulfate splitting from the natural substrates 3-sulfogalactocerebroside and ascorbyl-2-sulfate and the artificial substrate p-nitrocatecholsulfate, whereas galactose-6-sulfate, a substrate of galactose-6‑sulfurylase, was totally resistant. In contrast, leukocyte extracts of healthy donors were able to split sulfate also from galactose-6-sulfate. The ITP method allows therefore a rapid and simple differentiation between samples of MLD and MSD patients and healthy donors. Therefore, the isotachophoretic diagnostic assay from leukocyte extracts described here provides a fast and efficient way for the diagnosis of MLD and MSD patients and an elegant system to differentiate between these diseases in one assay.

Case report: a rare case of Hunter syndrome (type II mucopolysaccharidosis) in a girl

BackgroundHunter syndrome (mucopolysaccharidosis type II) is a recessive X-linked disorder due to mutations in the iduronate 2-sulfatase (IDS) gene. The IDS gene encodes a lysosomal enzyme, iduronate 2-sulfatase. The disease occurs almost exclusively in males. However, in the literature, 12 cases of the disease in females are known due to structural anomalies, a non-random chromosome X inactivation or chromosome X monosomy. The purpose of this article is to demonstrate a rare case of Hunter syndrome in a girl caused by a mutation in the IDS gene inherited from the mother and the presence of chromosome X of paternal origin, partially deleted in the long arm region - 46,X,del(X)(q22.1).Case presentationGirl M., 4 years old, entered the hospital with growth retardation, pain in the lower limbs, and joint stiffness, noted from the age of 18 months. After the karyotype analysis, which revealed a partial deletion of the long arm of chromosome X - 46, X, del (X) (q 22.1), Turner syndrome was diagnosed. However, due to the hurler-like facial phenotype, Hurler syndrome or type I mucopolysaccharidosis (MPS) was suspected. The study of lysosomal enzymes showed normal alpha-L-iduronidase activity and a sharp decrease in the activity of iduronate sulfatase in the blood: 0.001 μM/l/h, at a rate of 2.5–50 μM/l/h. Molecular genetic analysis revealed a hemizygous deletion in the IDS gene, which was not registered in the international Human Gene Mutation Database (HGMD) professional. This deletion was not detected in the girl’s father, but was detected in her mother in the heterozygous state.ConclusionsThus, the girl confirmed comorbidity - Turner syndrome with a partial deletion of the long arm of chromosome X of paternal origin, affecting the Xq28 region (localization of the IDS gene), and Hunter syndrome due to a deletion of the IDS gene inherited from the mother. The structural defect of chromosome X in the girl confirmed the hemizygous state due to the mutation in the IDS gene, which has led to the formation of the clinical phenotype of Hunter syndrome.

Electrochemical Quantification of Sulfatase Activity for Point-of-Care Diagnosis

Sulf 1&2, as the 6-O endosulfatase of heparan sulfate proteoglycans (HSPGs), has been reported in many previous works that it plays the critical role in the pathogenesis of a variety of human cancers, especially lung and breast cancer. However, there are very few reports about point-of-care detection of Sulf 1&2 currently. In this work, an electrochemical method using HSPGs as an anchor was employed to quantify the activity of Sulf 1&2. HSPGs, a glycoprotein with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains and is also a type of glycosaminoglycan, can mediate the activation of a variety of cell growth factor signal pathways to promote the progression of cancers. Here, electrochemical impedance spectroscopy (EIS) was used to monitor the changes of electrical properties of HSPGs with Sulf 1&2 variation. Through analyzing and calculating the electrical parameters obtained by EIS, such as charge, capacity and degree of phase, we established the calibration curve of sulf 1&2 concentration, which could be further used to evaluate the sulf 1&2 activity within human blood samples. This work offers a new method for sulf 1&2 detection and has great potential in the early detection of cancers.

SAT-553 Gut Microbiota Involved in Thyroxine Metabolism and Development of Subclinical Hypothyroidism

The diversity, structure, and stability of the gut microbiota can influence hosts’ nutrition, energy, metabolism, and immunity through intestinal nutrient-sensing mechanisms, the gut-brain axis, or changes in intestinal permeability. It was reported that gut microbiota can influence hosts’ selenium concentration and 3,5,3’-triiodothyronine (T3) conversion, therefore participating in thyroid hormone metabolism. The detailed relationship of microbiota and thyroid disorder is not clear yet. In our study, an open-level randomized clinical trail (RCT) was performed in 100 people with subclinical hypothyroidism (SCH) in July 2017. Patients were randomly divided into L-thyroxine treated group (dosage was depended on thyroid function test) or untreated group. Fecal sample were collected and microbiome were analyzed by 16S rDNA high-throughput sequencing. The discrepancy of the microbiome community structure were obvious between not only the two groups, but also individuals with different drug dose within L-thyroxine group, as calculated by principle components analysis (PCA), principle coordinate analysis (PCoA) and non-metric multi-dimensional scaling (NMDS). Moreover, the microbial profile of certain patients, who were diagnosed to need to adjust their drug dose after nine months (March, 2018), showed same tendencies in PCA and NMDS analysis, which means microbiota may have correlation with the development of SCH. The abundance of some species (eg., Bacteroides, Lactobacillus, Streptococcus) showed a positive correlation with the drug dose. Those species were reported with sulfatase or glucuronidase activities, which could participate in the hydrolysis process of thyroid hormone and secondary bile acid. These results indicated that microbiota attributed in thyroxine absorption process in gut, and cholesterol or bile acid level might have correlation with thyroid dysfunction. However, in our test, we did not observe the obvious correlation between drug dose and serum cholesterol level. Taken together, these results indicated that gut microbiota may act as an important factor in influencing the occurrence, development and prognosis of thyroid dysfunction, and may partially responsible for the high risk of SCH in patients with hypercholesterolemia. The casual relationship between gut microbiota and thyroid dysfunction need to be further discussing.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (βleavinggroupobs-Enz = -0.33) than the uncatalyzed reaction (βleavinggroupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (ΔβleavinggroupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

Expression, activity and localization of lysosomal sulfatases in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a leading cause of death world-wide. Recently, we showed that COPD is associated with gene polymorphisms in SUMF1, a master regulator of sulfatases. Sulfatases are involved in extracellular matrix remodeling and activated by SUMF1, but their role in the lung is poorly described. We aimed to examine how sulfatases are affected in the airways of patients with COPD compared to ever smokers and never smokers. We observed that mRNA expression of the sulfatases GALNS, GNS and IDS was increased, while protein expression of many sulfatases was decreased in COPD fibroblasts. Several sulfatases, including GALNS, IDS, and SGSH, showed increased activity in COPD fibroblasts. Examination of different sulfatases by immunofluorescence showed that IDS, ARSB, GNS and SGSH in fibroblasts were localized to sites other than their reported destination. Using a master panel from different organs, RNA expression of all sulfatases could be observed in lung tissue. Additionally, immunohistochemistry on lung biopsies indicated differing expression of sulfatases in COPD patients. In conclusion, mRNA, protein expression, sulfatase activity levels, and localization of sulfatases are altered in lung fibroblasts and lung tissue from COPD patients and may be mechanistically important in COPD pathogenesis. This could contribute to the understanding of the disease mechanism in COPD and in the long run, to lead to more individualized therapies.

Self-Referenced Ratiometric Detection of Sulfatase Activity with Dual-Emissive Urease-Encapsulated Gold Nanoclusters.

In this study, on the basis of the biomineralization capability of urease, a facile, one-step, and green synthetic method has been proposed for the fabrication of gold nanoclusters (AuNCs). The prepared urease-encapsulated AuNCs (U-AuNCs) exhibited strong red fluorescence emission (λem = 630 nm) with a quantum yield as high as 17%. Interestingly, at a low concentration, the U-AuNC solution was found to be a dual-emissive system with the blue emission of the dityrosine (diTyr) residues of urease and the red emission of the embedded AuNCs. Further experiments demonstrated that p-nitrophenol (PNP) can selectively suppress the 410 nm emission of the diTyr residues of U-AuNCs without affecting the red emission of the U-AuNCs. The fluorescence quenching mechanism between U-AuNCs and PNP was systematically studied, and the leading role of the inner filter effect (IFE) was identified. Additionally, based on the sulfatase-catalyzed hydrolysis of p-nitrophenyl sulfate (PNPS) to release PNP, a self-referenced ratiometric detection method for sulfatase, which plays a crucial role in sulfur cycling, degradation of sulfated glycosaminoglycans and glycolipids, and extracellular remodeling of sulfated glycosaminoglycans, was developed by using dual-emissive U-AuNCs as the signal readout, in which the diTyr residues served as the probe and the AuNCs functioned as the internal reference. This IFE-based ratiometric sensing strategy showed a good linear relationship over the range of 0.01-1 U/mL ( R2 = 0.997). The detection limit for sulfatase activity was 0.01 U/mL. The developed protocol was successfully used to detect sulfatase activity in human serum samples. The simplicity, rapidity, low cost, high credibility, good reproducibility, and excellent selectivity of the detection platform serve as an inspiration for further applications of fluorescent AuNCs in chemo/biosensing.

Dopamine-induced sulfatase and its regulator are required for Salmonella enterica serovar Typhimurium pathogenesis

Catecholamine hormones enhance the virulence of pathogenic bacteria. Studies in the 1980s made intriguing observations that catecholamines were required for induction of sulfatase activity in many enteric pathogens, including Salmonella enterica serovar Typhimurium. In this report, we show that STM3122 and STM3124, part of horizontally acquired Salmonella pathogenesis island 13, encode a catecholamine-induced sulfatase and its regulator, respectively. Induction of sulfatase activity was independent of the well-studied QseBC and QseEF two-component regulatory systems. S. Typhimurium 14028S mutants lacking STM3122 or STM3124 showed reduced virulence in zebrafish. Because catecholamines are inactivated by sulfation in the mammalian gut, S. Typhimurium could utilize CA-induced sulfatase to access free catecholamines for growth and virulence.

Enzymatic Hydrolysis of an Organic Sulfur Compound

Sulfatases which cleave sulfate esters in biological systems are key enzymes that deserve special attention due to their significant roles in organic sulfur (OS) mineralization and inorganic sulfur () release. In this study, in-vitro experiments were conducted to evaluate S bonded substrate hydrolysis by a commercially available arylsulfatase (EC 3.1.6.1) from Aerobacter aerogenes. The enzyme-substrate interactions were assessed to determine: 1) rate of hydrolysis, 2) catalytic efficiency, 3) thermal stability, and 4) optimal pH of this enzyme. Arylsulfatase exhibited substrate hydrolysis with a high affinity for p-nitrophenyl sulfate (potassium 4-nitrophenyl sulfate (pNPS)). The optimum activity for the enzyme was observed to occur at a pH of 7.1. The optimal temperature was 37°C but ranged from 35°C - 45°C. The apparent Km and Kcat of the enzyme for pNPS hydrolysis at the optimal pH, and temperature were determined to be 1.03 mM and 75.73 μM/min, respectively. This work defines the catalytic and kinetic properties of arylsulfatase (EC 3.1.6.1) and confirms the optimal conditions for sulfatase activity testing. The resulting information is useful in elucidating the contributions that individual enzymes have for specific reactions rather than relying on traditional total enzyme activity measurements.

One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the β-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

Responses of soil enzymatic activities to transgenic Bacillus thuringiensis (Bt) crops - A global meta-analysis

Transgenic Bacillus thuringiensis (Bt) crops have been widely planted, and the resulting environmental risks have attracted extensive attention. To foresee the impacts of Bt crops on soil quality, it is essential to understand how Bt crops alter the soil enzymatic activities and what the important influencing factors are. We compiled data from 41 published papers that studied soil enzymatic activities with Bt crops and their non-Bt counterparts. The results showed that dehydrogenase and urease significantly increased, but neutral phosphatase significantly decreased under Bt crop cultivations without Bt residues incorporation. The activities of dehydrogenase, β-glucosidase, urease, nitrate reductase, alkaline phosphatase, and aryl sulfatase significantly increased under Bt crop cultivation with Bt residues incorporation. The response ratios of other enzymes were not significantly changed. Generally, the response ratios of soil enzymes were greater with Bt residues incorporation than those of Bt crop cultivations without Bt residues incorporation. Further, the response ratios of soil enzymes varied with Bt crop types and growth periods. It was the strongest under Bt cotton among Bt crops, and the significant responses usually appeared in the middle growth stages. The responses of soil enzymes ascribed more to the properties of Bt crops than to soil properties across sites. Given - significant responses of some soil enzymes to Bt crops, we recommended that soil environmental risks should be carefully evaluated over the transgenic crops.

Access roads impact enzyme activities in boreal forested peatlands

We investigated the impacts of resource access roads on soil enzyme activities in contrasting forested boreal peatlands (bog and fen). In August 2016, a total of 72 peat samples were collected from twelve 20 m long transects perpendicular to access roads, with a further six samples collected from undisturbed reference areas. Sampling locations represent a range in three variables associated with roads: 1) side of the road (upstream/downstream), 2) distance to a culvert (longitudinal; <2 and >20 m), and 3) distance from the road (lateral; 2, 6, and 20 m). Phenol oxidase and hydrolase (glucosidase, sulfatase, xylosidase, glucosaminidase, and phosphatase) enzyme activities were determined for each sample, in addition to water table depth, phenolic concentration, pH, and peat temperature. The average hydrolase activities in the fen were ~four times higher than in the bog. At the bog, the water table depth, phenolic concentration, pH and the activities of phenol oxidase, sulfatase, glucosidase, xylosidase and glucosaminidase were all significantly influenced by one or more road associated factors. The highest enzyme activities in the bog occurred on the downstream side of the road at plots located far from the culvert. In contrast, the flow of water in the fen was not perpendicular to the road. Consequently, no significant variations in water table depth, phenolic concentration, pH or enzyme activity were found with respect to road associated factors. Results indicate that road crossings in boreal peatlands can indirectly alter enzyme activities, likely as part of a causal chain following changes to hydrology and redox conditions. Two of six investigated enzymes had significantly higher activities in the road disturbed areas compared to undisturbed areas, suggesting ultimately that roads may enhance organic matter decomposition rates. However, adequate hydrologic connections through culverts and road construction parallel to the water flow can minimize the road-induced impacts.

Recognition and ER Quality Control of Misfolded Formylglycine-Generating Enzyme by Protein Disulfide Isomerase

Multiple sulfatase deficiency (MSD) is a fatal, inherited lysosomal storage disorder characterized by reduced activities of all sulfatases in patients. Sulfatases require a unique post-translational modification of an active-site cysteine to formylglycine that is catalyzed by the formylglycine-generating enzyme (FGE). FGE mutations that affect intracellular protein stability determine residual enzyme activity and disease severity in MSD patients. Here, we show that protein disulfide isomerase (PDI) plays a pivotal role in the recognition and quality control of MSD-causing FGE variants. Overexpression of PDI reduces the residual activity of unstable FGE variants, whereas inhibition of PDI function rescues the residual activity of sulfatases in MSD fibroblasts. Mass spectrometric analysis of a PDI+FGE variant covalent complex allowed determination of the molecular signature for FGE recognition by PDI. Our findings highlight the role of PDI as a disease modifier in MSD, which may also be relevant for other ER-associated protein folding pathologies.