A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Mechanisms of arsenic hyperaccumulation in<Emphasis Type="Italic"> Pteris</Emphasis> species: root As influx and translocation

Several species of fern from the Pteris genus are able to accumulate extremely high concentrations of arsenic (As) in the fronds. We have conducted short-term unidirectional As influx and translocation experiments with ⁷³As-radiolabeled arsenate, and found that the concentration-dependent influx of arsenate into roots was significantly larger in two of these As-hyperaccumulating species, Pteris vittata (L.) and Pteris cretica cv. Mayii (L.), than in Nephrolepis exaltata (L.), a non-accumulating fern. The arsenate influx could be described by Michaelis-Menten kinetics and the kinetic parameter K _m was found to be lower in the Pteris species, indicating higher affinity of the transport protein for arsenate. Quantitative analysis of kinetic parameters showed that phosphate inhibited arsenate influx in a directly competitive manner, consistent with the hypothesis that arsenate enters plant roots on a phosphate-transport protein. The significantly augmented translocation of arsenic to the shoots that was seen in these As hyperaccumulator species is proposed to be due to a combination of the increased root influx and also decreased sequestration of As in the roots, as a larger fraction of As could be extracted from roots of the Pteris species than from roots of N. exaltata. This leaves a larger pool of mobile As available for translocation to the shoot, probably predominantly as arsenite.Abbreviations As ^V Arsenate - As ^III Arsenite - K _m Michaelis-Menten constant - P _i Phosphate - V _max Maximum rate of an enzyme-catalyzed reaction     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

NMR for direct determination of Km and Vmax of enzyme reactions based on the Lambert W function-analysis of progress curves

¹H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K_m and V_max, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8 mM. Using the Lambert W function the parameters K_m and V_max were fitted to obtain the experimental progress curve and resulted in K_m = 28 mM and V_max = 13 μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K_m = 379 μM and k_cat = 0.04 s^− 1. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

Arsenate, arsenite and dimethyl arsinic acid (DMA) uptake and tolerance in maize (Zea mays L.)

The influx of arsenate, arsenite and dimethyl arsinic acid (DMA) were studied in 7-day-old excised maize roots (Zea mays L.), and then related to arsenate, arsenite and DMA toxicity. Arsenate, arsenite and DMA influx was all found concentration dependent with significant genotypic differences for arsenite and DMA. Arsenate influx in phosphate starved plants best fitted the four-parameter Michaelis–Menten model corresponding to an additive high and low affinity uptake system, while the uptake of phosphate replete plants followed the two parameter model of Michaelis–Menten kinetics. Arsenite influx was well described by the two parameter model of ‘Michaelis–Menten’ kinetics. DMA influx was comprised of linear phase and a hyperbolic phase. DMA influx was much lower than that for arsenite and arsenate. Arsenate and DMA influx decreased when phosphate was given as a pre-treatment as opposed to phosphate starved plants. The +P treatment tended to decrease influx by 50% for arsenate while this figure was 90% for DMA. Arsenite influx increasing slightly at higher arsenite concentrations in P starved plants but at lower arsenite concentrations, there was little or no difference in arsenite uptake. Low toxicity was found for DMA on maize compared with arsenate and arsenite and the relative toxicity of arsenic species was As(V) > As(III) >> DMA.     

The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis

A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide (²H₂O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm⁻¹ and of the amide I band at 1605 cm⁻¹ were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients ε of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM⁻¹ cm⁻¹ at 1625, 1605, and 1365 cm⁻¹, respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V_max, K_m, and K_cat) determined by Lineweaver-Burk plot were 532.2  U mg⁻¹ protein, 6.4 mM, and 806.36 s⁻¹, respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.     

Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry

Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K_M) and maximum velocity (V_max) for both of the TcDHODH substrates: dihydroorotate (K_M = 8.6 ± 2.6 μM and V_max = 4.1 ± 0.7 μM s^-1) and fumarate (K_M = 120 ± 9 μM and V_max = 6.71 ± 0.15 μM s^-1). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K_M. Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.     

Interaction of arsenite with a zinc finger CCHC peptide: evidence for formation of an As-Zn-peptide mixed complex

The interaction of arsenite with a Cys₃His (CCHC) zinc finger model (34-51) HIV-1 nucleocapsid protein p7 (NCp7) peptide in the absence and presence of Zn^II was studied using fluorescence spectroscopy, CD (circular dichroism) and ESI-MS (Electrospray Ionization Mass Spectrometry). We found that arsenic forms different complexes with the free peptide and the zinc finger peptide. In the former case the peptide conformation differed greatly from that of the zinc finger, whereas in the second case a mixed As-Zn-peptide complex was formed with partial preservation of zinc finger conformation. An apparent stability constant was estimated for the mixed As-Zn-peptide complex (K = 2083 M^− 1 and 442 M^− 1 at 25 °C and pHs 6 and 7, respectively). Our study also shows that the interaction of arsenic with the CCHC motif is facilitated by glutathione (GSH), through formation of a GS-As-peptide conjugate.     

Triose phosphate isomerase from the blood fluke Schistosoma mansoni: Biochemical characterisation of a potential drug and vaccine target

The glycolytic enzyme triose phosphate isomerase from Schistosoma mansoni is a potential target for drugs and vaccines. Molecular modelling of the enzyme predicted that a Ser-Ala-Asp motif which is believed to be a helminth-specific epitope is exposed. The enzyme is dimeric (as judged by gel filtration and cross-linking), resistant to proteolysis and highly stable to thermal denaturation (melting temperature of 82.0 °C). The steady-state kinetic parameters are high (K_m for dihydroxyacetone phosphate is 0.51 mM; K_m for glyceraldehyde 3-phosphate is 1.1 mM; k_cat for dihydroxyacetone phosphate is 7800 s⁻¹ and k_cat for glyceraldehyde 3-phosphate is 6.9 s⁻¹).     

Accumulation, speciation, and coordination of arsenic in an inbred line and a wild type cultivar of the desert plant species Chilopsis linearis (Desert willow)

This study investigated the absorption of arsenic (As), sulfur (S), and phosphorus (P) in the desert plant Chilopsis linearis (Desert willow). A comparison between an inbred line (red flowered) and wild type (white flowered) plants was performed to look for differential responses to As treatment. One month old seedlings were treated for 7 days with arsenate (As₂O₅, As^V) at 0, 20, and 40 mg As^V L⁻¹. Results from the ICP-OES analysis showed that at 20 mg As^V L⁻¹, red flowered plants had 280 ± 11 and 98 ± 7 mg As kg⁻¹ dry wt in roots and stems, respectively, while white flowered plants had 196 ± 30 and 103 ± 13 mg As kg⁻¹ dry wt for roots and stems. At this treatment level, the concentration of As in leaves was below detection limits for both plants. In red flowered plants treated with 40 mg As^V L⁻¹, As was at 290 ± 77 and 151 ± 60 mg As kg⁻¹ in roots and stems, respectively, and not detected in leaves, whereas white flowered plants had 406 ± 36, 213 ± 12, and 177 ± 40 mg As kg⁻¹ in roots, stems, and leaves. The concentration of S increased in all As treated plants, while the concentration of P decreased in roots and stems of both types of plants and in leaves of red flowered plants. X-ray absorption spectroscopy analyses demonstrated partial reduction of arsenate to arsenite in the form of As-(SX)₃ species in both types of plants.     

Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with K_m = 0.38 mM and k_cat/K_m = 20.58 mM⁻¹ s⁻¹ for hydrolysis, and K_m2 = 18.38 mM and k_cat2/K_m2 = 2.57 mM⁻¹ s⁻¹ for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235—located on a wide open groove near subsite +1—is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.     

Dynamic synergistic effect on Trichoderma reesei cellulases by novel β-glucosidases from Taiwanese fungi

Dynamic synergistic effects in cellulosic bioconversion have been revealed between Trichoderma reesei cellulases and β-glucosidases (BGLs) from six Taiwanese fungi. A high level of synergy (8.9-fold) was observed with the addition of Chaetomellaraphigera BGL to T. reesei cellulases. In addition, the C. raphigera BGL possessed the highest activity (V_max/K_m = 46.6 U/mg mM) and lowest glucose inhibition (Ki = 4.6 mM) with the substrate 4-nitrophenyl β-d-glucopyranoside. For the natural cellobiose substrate, however, the previously isolated Aspergillus niger BGL Novo-188 had the highest V_max/K_m (0.72 U/mg mM) and lowest Ki (59.5 mM). The demonstrated dynamic synergistic effects between some BGLs and the T. reesei cellulase system suggest that BGLs not only prevent the inhibition by cellobiose, but also enhance activities of endo- and exo-cellulases in cellulosic bioconversion. Comparisons of kinetic parameters and synergism analyses between BGLs and T. reesei cellulases can be used for further optimization of the cellulosic bioconversion process.     

Characterization of glutathione reductase and catalase in the fronds of two Pteris ferns upon arsenic exposure

To better understand the mechanisms of plant tolerance to high concentration of arsenic, we characterized two antioxidant enzymes, glutathione reductase (GR) and catalase (CAT), in the fronds of Pteris vittata, an arsenic-hyperaccumulating fern, and Pteris ensiformis, an arsenic-sensitive fern. The induction, activation and apparent kinetics of GR and CAT in the plants upon arsenic exposure were investigated. Under arsenic exposure (sodium arsenate), CAT activity in P. vittata was increased by 1.5-fold, but GR activity was unchanged. Further, GR was not inhibited or activated by the arsenic in assays. No significant differences in K_m and V_max values of GR or CAT were observed between the two ferns. However, CAT activity in P. vittata was activated by 200 μM arsenate up to 300% compared to the control. Similar but much smaller increases were observed for P. ensiformis and purified bovine liver catalase (133% and 120%, respectively). This research reports, for the first time, the activation of CAT by arsenic in P. vittata. The increased CAT activities may allow P. vittata to more efficiently mediate arsenic-induced stress by preparing the fern for the impeding production of reactive oxygen species resulting from arsenate reduction to arsenite in the fronds.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

Toxicity, transformation and accumulation of inorganic arsenic species in a microalga Scenedesmus sp. isolated from soil

Arsenic speciation and cycling in the natural environment are highly impacted via biological processes. Since arsenic is ubiquitous in the environment, microorganisms have developed resistance mechanisms and detoxification pathways to overcome the arsenic toxicity. This study has evaluated the toxicity, transformation and accumulation of arsenic in a soil microalga Scenedesmus sp. The alga showed high tolerance to arsenite. The 72-h 50 % growth inhibitory concentrations (IC₅₀ values) of the alga exposed to arsenite and arsenate in low-phosphate growth medium were 196.5 and 20.6 mg? L^?1, respectively. When treated with up to 7.5 mg? L^?1 arsenite, Scenedesmus sp. oxidised all arsenite to arsenate in solution. However, only 50 % of the total arsenic remained in the solution while the rest was accumulated in the cells. Thus, this alga has accumulated arsenic as much as 606 and 761 μg? g^?1 dry weight when exposed to 750 μg? L^?1 arsenite and arsenate, respectively, for 8 days. To our knowledge, this is the first report of biotransformation of arsenic by a soil alga. The ability of this alga to oxidise arsenite and accumulate arsenic could be used in bioremediation of arsenic from contaminated water and soil.