scholarly journals The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures

BIO-PROTOCOL ◽  
2017 ◽  
Vol 7 (14) ◽  
Author(s):  
Patrick Rühl ◽  
Arnulf Kletzin
Keyword(s):  
High Temperatures ◽  
Elemental Sulfur ◽  
Reductase Activity ◽  
Activity Assay ◽  
Sulfur Oxygenase Reductase

Optimization of the nitrate reductase activity assay for citrus trees

2014 ◽  
Vol 37 (4) ◽  
pp. 383-390 ◽  
Author(s):  
Verónica Lorena Dovis ◽  
Franz Walter Rieger Hippler ◽  
Karina Iolanda Silva ◽  
Rafael Vasconcelos Ribeiro ◽  
Eduardo Caruso Machado ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Activity Assay ◽  
Citrus Trees

scholarly journals Constraints on Anaerobic Respiration in the Hyperthermophilic Archaea Pyrobaculum islandicum and Pyrobaculum aerophilum

2007 ◽  
Vol 74 (2) ◽  
pp. 396-402 ◽  
Author(s):  
Lawrence F. Feinberg ◽  
R. Srikanth ◽  
Richard W. Vachet ◽  
James F. Holden
Keyword(s):  
Iron Oxide ◽  
Growth Rates ◽  
Direct Contact ◽  
Iron Reduction ◽  
Elemental Sulfur ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Hyperthermophilic Archaea ◽  
Pyrobaculum Aerophilum ◽  
Pyrobaculum Islandicum

ABSTRACT Pyrobaculum islandicum uses iron, thiosulfate, and elemental sulfur for anaerobic respiration, while Pyrobaculum aerophilum uses iron and nitrate; however, the constraints on these processes and their physiological mechanisms for iron and sulfur reduction are not well understood. Growth rates on sulfur compounds are highest at pH 5 to 6 and highly reduced (<−420-mV) conditions, while growth rates on nitrate and iron are highest at pH 7 to 9 and more-oxidized (>−210-mV) conditions. Growth on iron expands the known pH range of growth for both organisms. P. islandicum differs from P. aerophilum in that it requires direct contact with insoluble iron oxide for growth, it did not produce any extracellular compounds when grown on insoluble iron, and it lacked 2,6-anthrahydroquinone disulfonate oxidase activity. Furthermore, iron reduction in P. islandicum appears to be completely independent of c-type cytochromes. Like that in P. aerophilum, NADH-dependent ferric reductase activity in P. islandicum increased significantly in iron-grown cultures relative to that in non-iron-grown cultures. Proteomic analyses showed that there were significant increases in the amounts of a putative membrane-bound thiosulfate reductase in P. islandicum cultures grown on thiosulfate relative to those in cultures grown on iron and elemental sulfur. This is the first evidence of this enzyme being used in either a hyperthermophile or an archaeon. Pyrobaculum arsenaticum and Pyrobaculum calidifontis also grew on Fe(III) citrate and insoluble iron oxide, but only P. arsenaticum could grow on insoluble iron without direct contact.

Insight into the Sulfur Metabolism by Thermoacidophilic Archaeon Metallosphaera cuprina with Genomic, Proteomic and Biochemical Tools

2015 ◽  
Vol 1130 ◽  
pp. 145-148 ◽  
Author(s):  
Jin Long Song ◽  
Cheng Ying Jiang ◽  
Shuang Jiang Liu
Keyword(s):  
Transfer Reaction ◽  
Elemental Sulfur ◽  
Hot Spring ◽  
Sulfur Metabolism ◽  
Genomic Data ◽  
Sulfur Oxidation ◽  
Low Grade ◽  
Hypothetical Model ◽  
Insight Into ◽  
Sulfur Oxygenase Reductase

Abstract. The thermoacidophilic archaeon Metallosphaeracuprina was isolated from a sulfuric hot spring. M. cuprina is able to oxidize elemental sulfur, tetrathionate (S4O62+) pyrite, and a range of low-grade ores, thus is attractive to biomining industry. Dissimilatory sulfur metabolism with a sulfur oxygenase reductase (SOR) system has been reported for members of Sulfolobus and Acidianus. But SOR system was not identified in the genome of M. cuprina. Recently, we have explored the sulfur metabolism of M. cuprina with genomic, proteomic, and biochemical tools. A hypothetical model of sulfur metabolism in M. cuprina was proposed on proteomic and genomic data, and proteins that involved in sulfur metabolism have been identified in our following studies. Specifically, DsrE/TusA homologs were biochemically characterized, and a novel thiosulfate transfer reaction was found during sulfur oxidation with M. cuprina. More recently, we cloned and identified a CoA-dependent NAD(P)H sulfur oxidoreductase from M.cuprina. The study will cover new understandings of the sulfur metabolism with M. cuprina.

Sulfur Oxygenase Reductase in Different Acidithiobacillus Caldus-Like Strains

2009 ◽  
Vol 71-73 ◽  
pp. 239-242 ◽  
Author(s):  
Claudia Janosch ◽  
Christian Thyssen ◽  
Mario A. Vera ◽  
Violaine Bonnefoy ◽  
Thore Rohwerder ◽  
...  
Keyword(s):  
Elemental Sulfur ◽  
Elevated Temperatures ◽  
Sulfur Oxidation ◽  
Aquifex Aeolicus ◽  
Acidithiobacillus Caldus ◽  
Acidophilic Bacteria ◽  
Cell Extracts ◽  
Moderate Thermophile ◽  
Acidianus Brierleyi ◽  
Sulfur Oxygenase Reductase

The elemental sulfur oxidising enzyme Sulfur Oxygenase Reductase (SOR) is very well investigated in acidothermophilic archaea, such as Acidianus brierleyi and Sulfolobus metallicus. In contrast, not much is known about the biochemistry of elemental sulfur oxidation in acidophilic bacteria. Recently, however, the SOR-encoding gene has been found also in a bacterial strain closely related to the moderate thermophile Acidithiobacillus caldus. Confusingly, for the latter species, also the involvement of the SOX system as well as thiosulfate:quinone oxidoreductase (TQO) and tetrathionate hydrolase (TTH) in sulfur compound oxidation has been proposed based on genome analysis. In this study, we have detected the sor-gene in other Acidithiobacillus caldus-like strains, isolated from various bioleaching habitats, indicating that SOR plays an important role in sulfur oxidation in this species. Based on sequence comparison, the new bacterial sor-genes are closely related and distant from the known archaeal sequences as well as from the SOR found in the neutrophilic bacterium Aquifex aeolicus. In addition, SOR activity has been detected in crude cell extracts from all Acidithiobacillus caldus-like strains tested. The enzyme is truly thermophilic as highest activities were achieved at 65 °C, which is far beyond the growth optimum of Acidithiobacillus caldus. This finding may give rise to the question whether the presence of SOR in Acidithiobacillus caldus is only relevant while growing at elevated temperatures. Currently, experiments are performed for testing this hypothesis (comparing growth and enzyme activities at 30 vs. 45 °C).

scholarly journals A calibration curve for immobilized dihydrofolate reductase activity assay

Data in Brief ◽  
2015 ◽  
Vol 4 ◽  
pp. 19-21 ◽  
Author(s):  
Priyanka Singh ◽  
Holly Morris ◽  
Alexei V. Tivanski ◽  
Amnon Kohen
Keyword(s):  
Dihydrofolate Reductase ◽  
Calibration Curve ◽  
Reductase Activity ◽  
Activity Assay

scholarly journals A Sulfur Oxygenase from the Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus with Atypically Low Reductase Activity

2016 ◽  
Vol 199 (4) ◽  
Author(s):  
Patrick Rühl ◽  
Uwe Pöll ◽  
Johannes Braun ◽  
Andreas Klingl ◽  
Arnulf Kletzin
Keyword(s):  
Active Site ◽  
Elemental Sulfur ◽  
Specific Activity ◽  
Reductase Activity ◽  
Sulfur Metabolism ◽  
Residual Activity ◽  
Site Directed Mutagenesis ◽  
Sequence Comparisons ◽  
Content Type ◽  
Denaturation Kinetics

ABSTRACT Sequence comparisons showed that the sulfur oxygenase reductase (SOR) of the haloalkaliphilic bacterium Thioalkalivibrio paradoxus Arh 1 (TpSOR) is branching deeply within dendrograms of these proteins (29 to 34% identity). A synthetic gene encoding TpSOR expressed in Escherichia coli resulted in a protein 14.7 ± 0.9 nm in diameter and an apparent molecular mass of 556 kDa. Sulfite and thiosulfate were formed from elemental sulfur in a temperature range of 10 to 98°C (optimum temperature ≈ 80°C) and a pH range of 6 to 11.5 (optimum pH ≈ 9; 308 ± 78 U/mg of protein). Sulfide formation had a maximum specific activity of 0.03 U/mg, or <1% of the corresponding activity of other SORs. Hence, reductase activity seems not to be an integral part of the reaction mechanism. TpSOR was most active at NaCl or glycine betaine concentrations of 0 to 1 M, although 0.2% of the maximal activity was detected even at 5 M NaCl and 4 M betaine. The melting point of TpSOR was close to 80°C, when monitored by circular dichroism spectroscopy or differential scanning fluorimetry; however, the denaturation kinetics were slow: 55% of the residual activity remained after 25 min of incubation at 80°C. Site-directed mutagenesis showed that the active-site residue Cys44 is essential for activity, whereas alanine mutants of the two other conserved cysteines retained about 0.5% residual activity. A model of the sulfur metabolism in T. paradoxus is discussed. IMPORTANCE Sulfur oxygenase reductases (SORs) are the only enzymes catalyzing an oxygen-dependent disproportionation of elemental sulfur and/or polysulfides to sulfite, thiosulfate, and hydrogen sulfide. SORs are known from mesophilic and extremophilic archaea and bacteria. All SORs seem to form highly thermostable 24-subunit hollow spheres. They carry a low-potential mononuclear nonheme iron in the active site and an indispensable cysteine; however, their exact reaction mechanisms are unknown. Typically, the reductase activity of SORs is in the range of 5 to 50% of the oxygenase activity, but mutagenesis studies had so far failed to identify residues crucial for the reductase reaction. We describe here the first SOR, which is almost devoid of the reductase reaction and which comes from a haloalkaliphilic bacterium.

scholarly journals Adaptation of nitrate reductase activity assay for high throughput screening of crops

2020 ◽  
Vol 49 (6) ◽  
pp. 23-33
Author(s):  
G I. Karlov ◽  
D. Y. Litvinov ◽  
P. N. Kharchenko ◽  
P. Yu. Krupin ◽  
S. Yu. Shirnin ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
High Throughput ◽  
Total Protein ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Potassium Nitrate ◽  
Activity Assay ◽  
Freeze Dried ◽  
The Difference ◽  
Nr Activity

The  possibility of freeze drying of plant material and its grinding in a shaking bead mill to determine the activity of nitrate reductase (NR) was studied. The effectiveness of applying this approach to high throughput mass screening of crops was shown. The assay was carried out on seedlings of common wheat (Triticum aestivum) of the following cultivars: Altigo, Vassa, Grom, Doka, Soberbash, Starshina, Fisht and spring wheat Novosibirskaya 67. The crops were grown during 4-5 weeks on substrate without nitrogen and on substrate supplemented with 50 millimol / l (mM) of potassium nitrate. Nitrate reductase in plants retained its activity after lyophilization and grinding of dried leaves in a mill. The proposed protocol for NR activity assay is suitable for plant lysates with an NR activity suffi cient to form nitrite in the range of 5–120 micromoles / l (μM) in 800 μl of reaction mix (for instance, freeze-dried sample originated from 100 mg of wheat seedling leaves).  Centrifugation of a plant lysate at 20,000 g almost did not change NR activity compared to 12,000 g that is achievable for most lab centrifuges. Lysates from fresh leaves contained signifi cantly more total protein than lysates from lyophilized leaves (with an equal amount of starting wet material). The difference in the nitrate-reducing activity in lysates from fresh and lyophilized leaves was not as high as the difference in protein concentration. Thus, the activity of NR calculated per g of total protein was higher in lyophilized leaves than in fresh leaves. The activity of NR was signifi cantly induced by nitrate for all cultivars. The basal and nitrate-induced NR activity varied widely between the cultivars, and the induction ranged from 2.5 fold for Novosibirskaya 67 variety and 2.7 fold for Vassa to 5.4 for Altigo and 5.7 fold for Grom.

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