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Microbial Biotechnology (17517915) 17(3)
Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacIQ/Ptrc system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L−1 upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL−1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g−1 benzaldehyde and 0.089 g 2-HPP g cell dry weight−1 h−1, respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633. © 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.
Scientific Reports (20452322) 14(1)
Avicennia marina is a salt-tolerance plant with high antioxidant and antibacterial potential. In the present work, a gene encoding MnSOD from Avicennia marina (AmSOD2) was cloned in the expression vectors pET28a. The resulting constructs were transformed into Escherichia coli strains Rosetta (DE3). Following the induction with Isopropyl β-d-1-thiogalactopyranoside, the protein His-AmSOD2 was expressed but dominantly found in the insoluble fraction of strain R-AmSOD2. Due to detection of mitochondrial transit peptide in the amino acid sequence of AmSOD2, the transit peptide was removed and AmSOD2 without transit peptide (tAmSOD2) was expressed in E. coli and dominantly found in the soluble fraction. The enzyme His-tAmSOD2 exhibited a molecular mass of 116 kDa in native condition. Nevertheless, in reducing conditions the molecular mass is 28 kDa indicating the enzyme His-tAmSOD2 is a tetramer protein. As shown by ICP analysis there is one mole Mn2+ in each monomer. The Pure His-tAmSOD2 was highly active in vitro, however the activity was almost three-fold lower than His-AmSOD1. Whereas the high stability of the recombinant His-AmSOD1was previously shown after incubation in a broad range pH and high temperature, His-tAmSOD2 was stable up to 50 °C and pH 6 for 1 h. The gene expression analysis showed that the gene encoding AmSOD2 is expressed in root, shoot and leaves of A. marina. In addition, the results show that the expression in the leaves was enhanced after treatment of plant with NaCl, H2O2, Cd2+ and Ni2+ indicating the important role of MnSOD in the resistant mechanism of mangroves. © 2024, The Author(s).
Microbial Cell Factories (14752859) 22(1)
Background: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. Results: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL− 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L− 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g− 1 DCW h− 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)–polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. Conclusions: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones. Graphical abstract: [Figure not available: see fulltext.]. © 2023, The Author(s).
Metallothioneins (MTs) are metal-binding proteins that have important roles in the homeostasis of heavy metals. In this study, the two MT genes was studied in response to phytohormones using the barley aleurone layer as a kind of model system. The aleurone layer was isolated from barley embryo-less half grains and was incubated for 24 h with different phytohormones. Based on the results the genes encoding HvMT2b2 and HvMT4 were down-regulated through gibberellic acid (GA), while they were and up-regulated through salicylic acid (SA). Despite this, these two genes were differentially expressed to other hormones. Furthermore, the proteins HvMT2b2 and HvMT4 were heterologous expressed as GST-fusion proteins in E. coli. The HvMT4 and HvMT2b2 heterologous expression in E. coli gives rise to 10- and 3-fold improvements in the accumulation capacity for Zn2+, respectively. Whereas the transgenic E. coli strain that expresses HvMT2b2 could accumulate Cd2+ three-fold higher than control. The expression of HvMT4 did not affect the accumulation of Cd2+. HvMT4 which is known as seed-specific isoform seems to be able to bind to Zn2+ with good affinity and cannot bind Cd2+. In comparison, HvMT2b2 was able to bind both Zn2+ and Cd2+. Therefore HvMT4 could serve a noteworthy role in zinc storage in barley seeds. The expression of HvMT4 is induced by SA 30-fold, concerning the untreated aleurone layer. Such results could provide good insights for the assessment of the effects of phytohormones in the molecular mechanism involved in essential metal storage in cereal seeds. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
Biometals (15728773) 35(5)pp. 889-901
The toxic nature of arsenic has left a trail of disastrous health consequences around the world. Microorganisms have developed various strategies to deal with arsenic. The presence of plasmid and chromosomal ars operons is one of the most important mechanisms for the detoxification of arsenic in bacteria. ArsR is a trans-acting regulatory protein and acts as a repressor on ars operon. The gene encoding ArsR from Corynebacterium glutamicum (CgArsR1) was cloned in expression vectors pET28a. The resulting constructs were transformed into Escherichia coli strains Rosetta (DE3) and Rosetta gami 2. Following the induction with Isopropyl β-d-1-thiogalactopyranoside, the protein His-CgArsR1 was found in the soluble fraction of strain Rg-CgArsR1. For comparison, ArsR from E. coli was also overexpressed in E. coli (strain Rosetta gami 2) as His-EcArsR. A strain containing empty vector pET28a was also used as a control strain. In the medium containing either arsenite (0.5 mM) or arsenate (0.5 mM), the strain Rg-CgArsR1 and Rg-EcArsR were able to accumulate 1200 and 700 µg/g DCW As3+, respectively. In comparison, the accumulation of As5+ in these strains was 338 and 232 µg/g DCW, respectively. Whereas both strains Rg-CgArsR1 and Rg-EcArsR were able to accumulate higher amounts of As3+ and As5+ with respect to control strain, the accumulation of arsenic in the strain Rg-CgArsR1 was significantly more efficient than strain Rg-EcArsR for removing As3+ and As5+. Based on the results the gene encoding CgArsR1 is a useful and efficient target gene for the modification of bacteria for bioremediation of arsenic from polluted soil and water. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
Phytochemistry (00319422) 187
Avicennia marina is a widely distributed mangrove species with high tolerance to salt, oxidative stress and heavy metals. In the preset work, we found that superoxide dismutase (SOD) activity increases in Avicennia marina leaves in response to salt and hydrogen peroxide. Monitoring the SOD using Western blot analysis revealed that the accumulation of SOD increased in response to hydrogen peroxide but not in response to salinity stress. Here we also isolated and cloned a gene encoding AmSOD1 which was classified into the group of plant CuZnSODs based on amino acid sequence analysis. AmSOD1 was heterologously expressed in the soluble fraction of E. coli strain Rosetta (DE3). The cells expressing His-AmSOD1 were more tolerant in response to hydrogen peroxide treatment but not salt stress, suggesting the involvement of AmSOD1 in hydrogen peroxide tolerance. The enzyme His-AmSOD1 exhibited a molecular mass of 38 kDa, but it could be monomer in reducing conditions indicating a double-strand protein with intra-molecular disulfide bridge. There are two copper and two zinc moles per mole of dimer form of His-AmSOD1 suggesting the binding of one copper and one zinc ions to each monomer. The Pure His-AmSOD1 was highly active in vitro and its activity was considerably enhanced when the growth medium of the cells producing AmSOD1 was supplemented with Cu2+. The high stability of the recombinant AmSOD1 after incubation in a broad range pH and high temperature is a distinctive feature for AmSOD1, which may open new insights for application of AmSOD1 as a protein drug in different medical purposes. © 2021 Elsevier Ltd
Ramezanpour, A. ,
Karami, K. ,
Kharaziha, M. ,
Zakariazadeh, M. ,
Lipkowski, J. ,
Shah piri, A. ,
Azizi, N. ,
Namazian, M. Polyhedron (02775387) 206
Throughout this study, a new palladium (II) complex, trans-[Pd(Naph)2Cl2], with naphazoline hydrochloride (Naphcon) as an imidazole derivative, 2-(naphthalen-1-ylmethyl)-4,5-dihydro-1H-imidazole;hydrochloride, was synthesized and characterized by elemental analysis, spectroscopic methods (UV–Vis, IR, and 1H NMR) and single crystal X-ray structure analysis. The cytotoxicity of Naphcon and the PdII complex were investigated in vitro against human breast (MCF-7) and cervical epithelial carcinoma (HeLa) cancer cells. The results revealed the higher anticancer activity of complex rather that of Naphcon against MCF-7 cell line. The findings of in vitro studies including fluorescence and UV–Vis spectroscopy, circular dichroism (CD), thermal denaturation and viscosity measurement indicated the interaction of the PdII complex with calf-thymus DNA (CT-DNA) via a combination of covalent and non-covalent interactions, whereas the free Naphcon interacted with CT-DNA mainly through the groove binding mode. Moreover, the ability of Naphcon and the PdII complex to cleave pUC57 plasmid DNA was investigated. In addition, the interaction of both Naphcon and its PdII complex was explored with bovine serum albumin (BSA) by means of absorption and fluorescence spectroscopy. The binding constant (Kb) could be calculated for compounds through these spectroscopic methods. Based on the competitive binding studies using Eosin, Ibuprofen and Digoxin as site markers, the binding site of the PdII complex and Naphcon was found to be located on site-III and I of BSA, respectively. Furthermore, protease activity of compounds was examined under physiological conditions. Finally, to validate all data obtained from biophysical studies, the molecular docking simulation was employed as a computational method. © 2021
Journal of Chemical Technology and Biotechnology (02682575) 96(5)pp. 1285-1291
BACKGROUND: Hexavalent chromium Cr(VI) is a toxic and carcinogenic heavy metal. It is highly water soluble and one of the most widely used metals in industry, resulting in soil and water contamination. Nowadays engineering microbes provide new strategies for remediation of heavy metals. RESULTS: In the present work, a gene encoding E. coli chromate reductase (YieF) was cloned in pCDF-1b and was transferred to both cells containing empty pET41a (control strain), and cells containing pET41a-OsMT1 (R-MT1) resulted in production of new strains R-YieF and R-YieF/MT1, respectively. Both proteins – YieF as His-tag fusion protein and OsMT1 as glutathione-S-transferase fusion protein – appeared in the soluble fraction of R-YieF/MT1 after induction with isopropyl-d-1-thiogalactopyranoside. The strain R-YieF/OsMT1 showed high tolerance to Cr6+ and accumulated significantly higher Cr6+ than R-MT1 (5 mg L−1), R-YieF (7 mg L−1) and control (2 mg L−1). The accumulation of Cr6+ was dependent on the initial concentration of Cr6+ in the medium, time and the presence of other toxic metals such as Ag+1, Hg+2 and Cd2+. CONCLUSION: This study demonstrated that the co-expression of YieF and OsMT1 could effectively enhance the accumulation of Cr(VI) by E. coli cells and opens new insights into bioremediation strategies. © 2020 Society of Chemical Industry. © 2020 Society of Chemical Industry
Karami, K. ,
Jamshidian, N. ,
Zakariazadeh, M. ,
Momtazi-borojeni, A. ,
Abdollahi, E. ,
Amirghofran, Z. ,
Shah piri, A. ,
Nasab, A.K. Computational Biology and Chemistry (14769271) 91
New palladium complexes with general formula trans-[Pd(L)2(OAc)2] (1,2), (L = Benzhydrazide and 2-Furoic hydrazide) have been synthesized and characterized with various methods including elemental analysis, FT-IR, 1HNMR and mass spectroscopy. Afterward their interactions with bovine serum albumin and calf thymus deoxyribonucleic acid have been investigated by UV–vis absorption, fluorescence emission and circular dichroism spectroscopy. Also, site‐selective replacement experiments with site probes have been carried out. Analysis of fluorescence spectrum indicated static quenching mechanism. Spectroscopic measurements for DNA binding showed the groove binding to DNA for both complexes. Furthermore, cytotoxicity studies of complexes and cis-platin have been done against colon carcinoma (CT26) and breast cancer (4T1) cell lines. Evaluation of complexes (1) and (2) on induction of apoptosis in CT26 cells has been done. Finally, plasmid cleavage ability of (1) and (2) was investigated by gel electrophoresis that indicate the more activity of (1) than (2). © 2021 Elsevier Ltd
Environmental and Experimental Botany (00988472) 187
Arsenic is the most prevalent environmental toxic element and causes a variety health effects. Phytochelatins (PCs) are naturally occurring peptides with high affinity toward metal(loid)s that are present in plants and fungi. Here, the gene encoding phytochelatin synthase from Helianthus annuus (HaPCS) was isolated and cloned in expression vectors pET28a and pET41a. The resulting constructs were transformed into Escherichia coli strains Rosetta (DE3) and Rosetta gami 2. Following the induction with Isopropyl β-D-1-thiogalactopyranoside, the enzyme GST-HaPCS was expressed in the strains Rg-41a-HaPCS and R-41a-HaPCS and the enzyme His-HaPCS was expressed in R-28a-HaPCS. The comparison of soluble and insoluble extracted fractions revealed that the expression of GST-HaPCS in the strain Rg-41a-HaPCS is the optimum method for expression of this enzyme. The recombinant and pure GST-HaPCS catalysed PCs in vitro only in the presence of Cd2+, As3+ and As5+. The presence of Cd2+ activated the enzyme GST-HaPCS more than As3+/As5+. Therefore the enzyme GST-HaPCS seems to be differentially activated by different metal(loid)s. The strain Rg-41a-PCS was able to synthesise PCs when the medium was supplemented by As3+, As5+ and Cd2+. In these conditions the new strains were able to accumulate 10.67 μmol/g DCW As5+, 7.58 μmol/g DCW As3+ and 25.97 and 25.97 μmol/g DCW in medium containing 0.5 mM of each of these metal(loid)s. Furthermore in the present work we found that there was no difference between the expressions of HaPCS in H. annuus in response to different metal(loid)s. Therefore the higher accumulation of PCs and subsequently higher accumulation of Cd2+ could be due to the higher activity of HaPCS in the presence of this metal ion. This study introduces a good candidate gene from plant source with high ability for synthesis of PCs and accumulation of arsenic and cadmium. © 2021 Elsevier B.V.
Phytochemistry (00319422) 176
Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins that are important for essential metal homeostasis, protection against oxidative stress, and buffering against toxic heavy metals. In this work the gene encoding an MT type 2 from Avicennia marina (Forssk.) Vierh. (AmMT2) was cloned into pET41a and transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl β-D-1-thiogalactopyranoside, AmMT2 was expressed as glutathione-S-transferase (GST)-tagged fusion protein. The accumulation of Zn2+, Cu2+, Fe2+, Ni2+ and Cd2+ for strain R-AmMT2 was 4, 8, 5.4, 2 and 1.6 fold of control strain suggesting the role of AmMT2 in accumulation of metals. Particularly the strain R-AmMT2 was able to accumulate 30.7 mg per g dry weight. The cells expressing AmMT2 was more tolerant to hydrogen peroxide and had higher catalase (CAT) activity. To understand the mechanistic action of AmMT2 hydrogen peroxide tolerance, the activity of CAT in the E. coli protein extract was assayed after addition of pure Fe2+/GST-AmMT complex and Apo/GST-AmMT2 in vitro. Whereas, the activity of CAT did not change by the addition of Apo/GST-AmMT2, the activity of CAT significantly increased after addition of Fe2+/GST-AmMT2. These results show that AmMT2 activates CAT through Fe2+ transfer which subsequently causes the oxidative stress tolerance. © 2020 Elsevier Ltd
Alinaghi, M. ,
Karami, K. ,
Shah piri, A. ,
Nasab, A.K. ,
Momtazi-borojeni, A. ,
Abdollahi, E. ,
Lipkowski, J. Journal of Molecular Structure (00222860) 1219
A novel Pd(II) complex, [Pd(C6H5N2O)2], containing pyridine-2-carbaldehyde oxime ligand has been synthesized and characterized using elemental analysis, Fourier-transform infrared, nuclear magnetic resonance and mass spectroscopy. The single crystal structure of this Pd(II) complex has been determined by X-ray crystallography. Based on the DNA binding studies including ultraviolet visible spectrophotometry, fluorescence emission titration and viscosity measurement, the interaction of Pd(II) complex with calf thymus DNA occurs by groove binding. In the absence of an external reductant, the Pd(II) complex cleaves the supercoiled double-stranded DNA under physiological conditions. Moreover, in the presence of Pd(II) complex, the Bovine Serum Albumin microenvironment and secondary structure change. On the basis of the competitive experiments using site markers, the complex is mainly located in site I of the protein. The binding of the Pd(II) complex to DNA was modeled using molecular docking. The antitumor impacts of the ligand and the Pd(II) complex were evaluated in vitro against the mouse colon carcinoma (C26) and melanoma (B16–F0) tumor cell lines. The antitumor activity has been significantly improved by the complexation process. IC50 values smaller than those of cisplatin have been shown by the Pd(II) complex and oxime ligand against cancer cell lines. In addition, Pd(II) complex has been tested against NIH normal fibroblast cells. Consequently, Pd(II) complex may be considered a selective compound against cancer cells, according to the SI definition. © 2020
Protein And Peptide Letters (09298665) 27(10)pp. 945-952
Background: The replacement of carbohydrate sweeteners with protein sweeteners from plants has attracted the interest of researchers because these proteins don’t trigger the insulin response and are more nutritive for consumption in food. Brazzein (Braz) is a small and heat-stable sweet protein that has been originally derived from African plant Pentadiplandra brazzeana. In the present work the solubility, sweetness and yield of recombinant forms of Braz in two expression hosts, E. coli and S. cerevisiae were comprised. Methods: The codon-optimized gene of Braz was cloned in expression vectors pET28a and pET41a and GPD. The resulted vectors pET28a-Braz and pEt41a-Braz were transformed into Escherichia coli strain Rosetta (DE3) and the vector GPD-Braz was transformd to S. cerevisiae. The expression of Braz in different systems was analyzed by SDS-PAGE and western blotting. Results: The results verified the heterologous expression of Braz in S. cerevisiae carrying GPD-Braz. Also the expression of Braz as carboxy-terminal extensions of His-tag and Glutathione-S-Transferase (GST) were verified in transgenic E. coli containing pET28a-Braz and pET41a-Braz, respectively. Conclusion: Although the yield of GST-Braz was higher than His-Braz and Braz expressed in S. cerevisiae, but the higher solubility, sweetness, safety (GRAS) are important advantages of the use of S. cerevisiae as expression host for production of Braz. Therefore the result of present work opens new insights for providing the new sweet yeasts that can be used as food additives. © 2020, Bentham Science Publishers. All rights reserved.
Protein Expression and Purification (10960279) 175
The simultaneously functions of Metallothioneins (MTs) are relied on their metalation mechanisms that can be divided into non-cooperative, weakly cooperative and strongly cooperative mechanisms. In this study, we recombinantly synthesized OsMTI-1b, N- and C-terminal Cys-rich regions as glutathione-S-transferase (GST)-fusion proteins in E. coli. In comparison with control strains (The E. coli cells containing pET41a without gene), transgenic E. coli cells showed more tolerance against Cd2+ and Zn2+. The recombinant GST-proteins were purified using affinity chromatography. According to in vitro assays, the recombinant proteins showed a higher binding ability to Cd2+ and Zn2+. However, the affinity of apo-proteins to Cu2+ ions were very low. The coordination of Cd2+ ions in OsMTI-1b demonstrates a strongly cooperative mechanism with a priority for the C-terminal Cys-rich region that indicates the detoxifying of heavy metals as main role of P1 subfamily of MTs. While the metalation with Zn2+ conformed to a weakly cooperative mechanism with a specificity to N-terminal Cys-rich region. It implies the specific function of OsMTI-1b is involved in zinc homeostasis. Nevertheless, a non-cooperative metalation mechanism was perceived for Cu2+ that suggests the fully metalation does not occur and OsMTI-1b cannot play a significant role in dealing with Cu2+ ions. © 2020 Elsevier Inc.
Karami, K. ,
Jamshidian, N. ,
Bagheri, A. ,
Hajiaghasi, A. ,
Momtazi-borojeni, A. ,
Abdollahi, E. ,
Shah piri, A. ,
Azizi, N. ,
Lipkowski, J. Journal of Molecular Structure (00222860) 1206
The new alkoxime ligand (E)-anthracene-9-carbaldehyde-O-methyl oxime (1) derivative of 9-anthraldehyde and its palladium (II) complexes trans-Pd(C15H10NOCH3)2(OAc)2 (2) and [cis-Pd(C15H10NOCH3)(Py)2(OAc)](OAc) (3) were synthesized and characterized by spectroscopic techniques. Also, the ligand (1) structure was characterized by X-ray crystallography. Then their interaction with calf thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) was explored using absorption and emission spectral methods. The inner filter effect was corrected to get accurate data. Ligand (1) and complex (3) bind strongly to DNA via intercalation and complex (2) binds to it by groove binding mode. The binding modes were further confirmed by CD spectra. BSA binding aspects of synthesized compounds have been studied through various biophysical techniques, and the efficiency of energy transfer between them and BSA has been calculated according to Förster energy transfer theory (FRET). Furthermore, they were assessed for in vitro cytotoxicity testing on the colon carcinoma (CT26) and breast cancer (4T1) cell lines. The three compounds displayed better cytotoxic profiles against CT26 than 4T1 cancer cell lines. Also, their ability to cleave PUC57 plasmid DNA was investigated. Finally, the interaction of ligand (1) with DNA and BSA has been substantiated through molecular docking and a promising intercalation binding efficacy has been revealed for it. © 2019
Applied Biochemistry and Biotechnology (02732289) 187(2)pp. 649-661
β-Lactoglobulin (BLG), a member of lipocalin family, is one of the major bovine milk allergens. This protein exists as a dimer of two identical subunits and contains two intramolecular disulfide bonds that are responsible for its resistance to trypsin digestion and allergenicity. This study aimed to evaluate the effect of reduction of disulfide bonds of BLG with different rice thioredoxins (Trxs) on its digestibility and allergenicity. Therefore, the active recombinant forms of three rice Trx isoforms (OsTrx1, OsTrx20, and OsTrx23) and one rice NADPH-dependent Trx reductase isoform (OsNTRB) were expressed in Escherichia coli. Based on SDS-PAGE, HPLC analysis, and competitive ELISA, the reduction of disulfide bonds of BLG with OsNTRB/OsTrx23, OsNTRB/OsTrx1, GSH/OsTrx1, or GSH/OsTrx20 increased its trypsin digestibility and reduced its immunoreactivity. The finding of this study opens new insights for application of plant Trxs in the improvement of food protein digestibility. Especially, the use of OsTrx20 and OsTrx1 are more cost-effective than E. coli and animal Trxs due to their reduction by GSH and no need to NADPH and Trx reductase as mediator enzyme. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.
Protein And Peptide Letters (09298665) 26(10)pp. 768-775
Introduction: Metallothioneins (MTs) are members of a family of low molecular weight and cysteine-rich proteins that are involved in heavy metal homeostasis and detoxification in living organisms. Plants have multiple MT types that are generally divided into four subgroups according to the arrangement of Cys residues. Methods: In the present study the E. coli cells which heterologously express four different rice MT (OsMT) isoforms were analyzed for the accumulation of two forms of chromium, Cr3+ and Cr6+. Results: The results show that the transgenic bacteria were more tolerant than control cells when they were grown up in the medium comprising Cr(NO3)3.9H2O or Na2CrO4. The cells expressing OsMT1, OsMT2, OsMT3 and OsMT4 give rise to 6.5-, 2.7-, 5.5-and 2.1-fold improvements on the accumulation capacity for Cr3+ and 9-, 3-, 5-and 3-fold Cr6+ respectively compared with comparison to the control strain. Furthermore, the purified recombinant GST-OsMTs were tested for their binding ability to Cr+3 and Cr+6 in vitro. Discussion: The data show that the recombinant GST-OsMT1 and GST-OsMT2 were able to bind both Cr3+ and Cr6+, in vitro. However, their binding strength was low with respect to previous tested divalent ions like Cd2+. © 2019 Bentham Science Publishers.
Protein And Peptide Letters (09298665) 26(3)pp. 176-183
Background: Acetohydroxyacid Synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched chain amino acids. AHAS is the common target site of five herbicide chemical groups: sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone Objective: The purification of protein enabled us to study the physical and biochemical properties of the enzyme. In addition in vitro activity of this enzyme was tested in the presence of four different sulfonylureaherbicides and the feedback regulation of enzyme was analyzed in the presence of branched amino acids. Methods: The gene encoding catalytic subunit of rice AHAS (cOsAHAS) without part of the chloroplast transit sequence was cloned into the bacterial expression vector pET41a and heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST).The soluble protein was purified using affinity chromatography. The measurement of GSTOsAHAS activity was performed under optimized conditions at present of branched-chain amino acids and sulfonylurea herbicides independently. Results: The optimum pH and temperature for GST-cOsAHAS activity was 8.0 and 37 °C, respectively. The specific activity and Km value of this enzyme toward pyruvate were 0.08 U/mg and 30 mM, respectively.GST-cOsAHAS was inhibited by herbicides tribenuron, sulfosulfuron, nicosulfuron and bensulfuron while the enzyme was insensitivieto end products. Conclusion: These results suggest that the recombinant form of GST-cOsAHAS is functionally active and carries the binding site for sulfynylurea herbicides. Furthermore, GST-cOsAHAS was insensitive to feedback inhibition by endproducts which indicates the existence of a regulator subunit in rice AHAS as previously has been described in other plant AHASs. © 2019 Bentham Science Publishers.
Karami, K. ,
Ramezanpour, A. ,
Zakariazadeh, M. ,
Shah piri, A. ,
Kharaziha, M. ,
Kazeminasab, A. ChemistrySelect (23656549) 4(17)pp. 5126-5137
One of the most active areas within the field of bioorganometallic chemistry, complexes of N-heterocyclic carbenes (NHCs), have recently gained interest. Herein, we report two luminescent palladium N-heterocyclic carbene complexes; namely [Pd{(C,N)-C 6 H 4 CH 2 NH(CH 2 CH 3 )}(1)] (2) and [Pd{(C,N)-C 6 H 4 CH 2 NH 2 }(1)] (3) (1=1-methyl-3-(2-oxo-2-(pyren-1-yl)ethyl)-2,3-dihydroimidazol-2-ylidene) which were synthesized from the reaction of luminescent imidazolium salt (1(H)Br) and binuclear Palladacycles. The interactions of them with CT-DNA evaluated via absorption, emission and CD spectral techniques as well as measurements of viscosity and thermal denaturation and the results have been shown that they bounded to CT-DNA by intercalation and groove binding modes. The in vitro cytotoxicity of compounds 2–3 and 1(H)Br on human breast (MCF-7) and cervical epithelial carcinoma (HeLa) cancer cells lines, indicated the wide range of anticancer activities of them with low IC 50 values. Moreover, based on the protein binding ability studies, the intrinsic fluorescence of BSA could be strongly quenched by compounds via a static quenching mechanism. Competitive binding study using Eosin, Digoxin and Ibuprofen as site markers, indicated that the compounds could bind to sites I and II on BSA structure. Finally, all data obtained from biophysical studies were validated by molecular modeling study. Computational results showed that palladium complexes have the potential for detection of mismatch DNA. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Current Microbiology (03438651) 75(11)pp. 1537-1542
Metallothioneins (MTs) are low-molecular weight cysteine (Cys)-rich proteins with high metal-binding capacity. Based on the Cys arrangement in their amino acid sequences, plant MTs are categorized into four classes. This study evaluated the ability of genetically engineered Escherichia coli cells, which express four rice MT isoforms as fusion proteins with glutathione-S-transferase (GST), to remove arsenic. As compared with control strain, the expression of GST-OsMT1, GST-OsMT2, GST-OsMT3, and GST-OsMT4 resulted in 8-, 5.6-, 3-, and 1.1-fold-higher As3+ accumulation. The recombinant GST-OsMT isoforms were purified using affinity chromatography and their apo-forms were prepared. The ability of the GST-OsMT2 isoform to bind with As3+ in vitro was also confirmed by ultraviolet (UV) absorption spectra recorded after the reconstitution of apo-proteins with As3+. However, the formation of complexes of other MT isoforms with arsenic was not observed. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.
Annals of Microbiology (18692044) 68(3)pp. 145-152
Mercury is one of the more common and potentially most harmful toxic metals. Remediation using conventional physical and chemical methods is uneconomical and generates large volumes of chemical waste. Bioremediation of hazardous metals has received considerable and growing interest over the years. In the present work, genetically engineered Escherichia coli cells, which express four rice metallothionein (MT) isoforms as fusions with glutathione-S-transferase (GST), were tested for their ability to remove mercury. The results showed that the E. coli cells expressing OsMT1, OsMT2, OsMT3, and OsMT4 are able to remove 20, 13.7, 10, and 7 nmol Hg2+/mg (dry weight) from the culture medium, respectively. The recombinant GST–OsMTs were purified using affinity chromatography. The UV absorption spectra and the results of 5,5-dithio-bis-(2-nitrobenzoic) acid (DTNB) assay recorded after the reconstitution of the apo-OsMTs with mercury confirmed that the different OsMT isoforms were able to form mercury complexes in vitro with different binding capacities and different binding strength. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan.
Biocatalysis and Agricultural Biotechnology (18788181) 9pp. 174-176
The aleurone layer plays a key role in germination by responding to hormone signals from embryo and producing hydrolases. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signaling molecules in an isolated system. In this work, the effect of ethephon on the protein release from gibberellic acid (GA)-treated aleurone layer was presented for two important enzymes in starch degradation: α-amylase and limit dextrinase (LD). The results showed that the release of proteins from aleurone layer to culture supernatant was remarkably increased when GA- treated aleurone layer was exposed to 1000–10000 ppm ethephon. Ethephon enhanced the release of α-amylase and advanced the release time of LD from GA-treated aleurone layer to culture supernatant. The results supports that the release of LD is dependent to cell wall degradation which can be induced by the effect of ethephon. © 2016 Elsevier Ltd
Brazilian Journal Of Microbiology (15178382) 48(3)pp. 537-543
Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd2+, H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd2+ and accumulated more Cd2+ ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses. © 2017 Sociedade Brasileira de Microbiologia
International Journal of Biological Macromolecules (01418130) 96pp. 436-441
In this study we examined the independent self assembly of metal-binding in C-terminal Cys- rich region of a type 1 metallothionein (MT) isoform from rice (OsMTI-1b). To this end the N-terminal of OsMTI-1b (C-OsMTI-1b) was heterologously expressed in Escherichia coli as fusion protein with glutathione-S-transferase (GST). As compared with control (The E. coli cells containing pET41a without gene), transgenic E. coli cells expressing GST-C-OsMTI-1b accumulated more Ni2+, Cd2+, and Zn2+ from culture medium and showed increased tolerance against these metals. The recombinant GST-C-OsMTI-1b was purified using affinity chromatography. According to in vitro assays the protein GST-C-OsMTI-1b was able to form complexes with Ni2+, Cd2+ and Zn2+. These results demonstrate the formation of independent metal-thiolate cluster at C-terminal Cys-rich region of OsMTI-1b without participation of N-terminal Cys-rich region. © 2016 Elsevier B.V.
Molecular Biology Research Communications (2322181X) 6(1)pp. 33-40
Thioredoxins (Trxs) are small ubiquitous oxidoreductase proteins with two redox-active Cys residues in a conserved active site (WCG/PPC) that regulate numerous target proteins via thiol/disulfide exchanges in the cells of prokaryotes and eukaryotes. The isoforms OsTrx23 with a typical active site (WCGPC) and OsTrx20 with an atypical active site (WCTPC) are two Trx h- type isoforms in rice that were previously found to be reduced by NADPH-dependent thioredoxin reductase and GSH/Grx system, respectively. In the present work the reduction of mutants G41TOsTrx23, T55GOsTrx20, K48DOsTrx20 and T55G-K48D OsTrx20 as well as wild types OsTrx23 and OsTrx20 were tested in the reaction containing either NADPH/NTR or glutathione (GSH). The results revealed that reduction rate of T55GOsTrx20 was remarkably decreased by GSH as compared to WtOsTrx20 highlighting the critical role of Thr-55 in interaction of OsTrx20 with GSH. On the other hand a significant decrease in the reduction rate of G41TOsTrx23 was observed in reaction containing NADPH-dependent thioredoxin reductase as compared with readuction rate of WtOsTrx23. These results suggest that first residue after N-terminal active site Cys is one of the critical residue in determination of system that Trxs can be reduced in.
New Journal of Chemistry (11440546) 40(11)pp. 9081-9097
A new Pd(ii) complex, [Pd(4-OHPh-tpy)Cl]Cl, with a ligand that is a terpyridine derivative, (4′-(4-hydroxyphenyl)-2,2′,6′,2′′-terpyridine (4-OHPh-tpy)), was prepared and fully characterized. The single crystal structures of 4-OHPh-tpy and its Pd(ii) complex were determined by X-ray crystallography. The in vitro studies (UV-Vis spectroscopy, circular dichroism (CD), rheometry, and gel electrophoresis) show that the Pd(ii) complex interacts with calf-thymus DNA (CT-DNA) via a combination of covalent, intercalation, and hydrogen bonding interactions, whereas the free 4-OHPh-tpy ligand interacts with DNA mainly through intercalation. The Pd(ii) complex cleaves the supercoiled double-stranded DNA under physiological conditions without the need to add an external reductant. The microenvironment and the secondary structure of BSA were also changed in the presence of both the free ligand and the Pd(ii) complex. The Pd(ii) complex was remarkable in exhibiting cleavage of the BSA at micromolar concentrations and short incubation times at physiological pH and temperature. The anti-cancer effects of the free ligand and the Pd(ii) complex were tested in vitro against five human tumor cell lines, including the human breast cancer cell line (MCF-7), lung adenocarcinoma cells (A-549), an erythroleukemic cell line (K562), a colorectal adenocarcinoma cell line (HT-29), and hepatocellular carcinoma (Hep-G2) cells. The Pd(ii) complex showed the IC50 values less than those of cisplatin against the MCF-7, A-549, K562, and HT-29 cancer cell lines. Finally, the binding of the Pd(ii) complex and 4-OHPh-tpy to BSA and DNA was modeled by molecular docking and molecular dynamic simulation methods. © 2016 The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.
International Journal of Biological Macromolecules (01418130) 88pp. 491-496
Metallothioneins (MTs) are a family of Cys-rich, low molecular weight, cytoplasmic metal binding proteins. MTs are present in all eukaryotes as well as some prokaryotes. Plant MTs are divided into four types based on Cys distribution pattern in their amino acid sequences. In the present work, the gene encoding OsMTI-2b, a type 2 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into Escherichia coli strain Rosetta (DE3). Following the induction with Isopropyl β-d-1-thiogalactopyranoside the OsMTI-2b was expressed as carboxyl-terminal extensions of glutathione-S-transferase (GST-tag), a 6His-tag, and an S-tag. The expressed recombinant fusion protein was named GST-OsMTI-2b. As compared with control, transgenic E. coli cells expressing GST-OsMTI-2b accumulated more Pb2+, Ni2+, Cd2+, Zn2+ and Cu2+ from culture medium and showed increased tolerance against these metals. Furthermore the E. coli cells expressing OsMTI-2b accumulated significantly higher Pb2+ than previously made strains which expressing other rice OsMT isoforms. The recombinant GST-OsMTI-2b was purified using affinity chromatography. According to in vitro assays the protein GST-OsMTI-2b was able to form complexes with Pb2+, Ni2+, Cd2+ and Zn2+. However, the binding ability for the different metals differed in the order: Pb2+ > Cd2+ > Zn2+ > Ni2+. © 2016 Elsevier B.V.
Biotechnology Letters (15736776) 38(3)pp. 503-508
Objectives: To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool. Results: Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l−1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l−1. Conclusions: Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast. © 2015, Springer Science+Business Media Dordrecht.
Zarei, M. ,
Shah piri, A. ,
Esmaeilnejad-ahranjani, P. ,
Arpanaei, A. RSC Advances (20462069) 6(52)pp. 46785-46793
In this contribution, magnetic nanocomposite particles of Fe3O4 cluster@SiO2 (MNPs) were functionalized with N-(2-aminoethyl)-3-aminopropyltrimethoxy-silane (EDS) to obtain amine-functionalized magnetic nanocomposite particles (AF-MNPs). Then, a novel nanobiohybrid magnetic adsorbent was developed by immobilization of the recombinant form of a rice metallothionein (MT) isoform onto the surface of the synthesized AF-MNP (MT-AF-MNPs) for the highly efficient removal of cadmium ions from aqueous media. The kinetic data for both AF-MNPs and MT-AF-MNPs were best fitted to the pseudo-second-order kinetic model. The best interpretation for the equilibrium data was given by a Langmuir isotherm, and the maximum cadmium adsorption capacities for AF-MNPs and MT-AF-MNPs were 320 mg g-1 and 476 mg g-1, respectively. The adsorption process for both nanocomposite particles was pH dependent and the adsorption capacity was found to decrease with decreasing of the pH. The cadmium-loaded adsorbents could be recovered readily by magnetic separation and regenerated easily by acid treatment. The continuous adsorption-desorption studies indicated that the AF-MNPs and MT-AF-MNPs retain 70% and 56% of their initial adsorption capacity after four adsorption-desorption cycles, respectively. The findings of the present work show the great potential of MT-AF-MNPs as an effective and recyclable adsorbent for the removal of cadmium ions from waste water. © The Royal Society of Chemistry 2016.
Protein And Peptide Letters (09298665) 23(7)pp. 639-644
The members of plant metallothionein (MT) subfamily p1 are characterized with the presence of six Cys at each end of N- and C-terminal of their amino acid sequences which are arranged in a CXCXXXCXCXXXCXC and CXCXXXCXCXXCXC sequence, respectively. In this study we evaluated the independence of N-terminal Cys-rich region of a type 1 MT isoform from rice (OsMTI-1b) in forming metal-thiolate cluster. To this end the N-terminal of OsMTI-1b (N-OsMTI-1b) was heterologously expressed in Escherichia coli as fusion protein with glutathione-S-transferase (GST). The E.coli cells expressing GST-N-OsMTI-1b were able to remove Cd2+ and Ni2+ from culture medium. The recombinant GST-N-OsMTI-1b was purified using affinity chromatography. The UV absorption spectra recorded after the reconstitution of the apo-protein with Cd2+ and Ni2+ confirmed that GST-N-OsMTI-1b was able to form complexes with Cd2+ and Ni2+ . These results demonstrate the formation of independent metal-thiolate cluster at N-terminal Cys-rich region of GST-N-OsMTI-1b without participation of C-terminal Cys-rich region. © 2016 Bentham Science Publishers.
Hägglund, P. ,
Finnie, C. ,
Yano, H. ,
Shah piri, A. ,
Buchanan, B.B. ,
Henriksen, A. ,
Svensson, B. Biochimica et Biophysica Acta - Proteins and Proteomics (15709639) 1864(8)pp. 974-982
Thioredoxins are nearly ubiquitous disulfide reductases involved in a wide range of biochemical pathways in various biological systems, and also implicated in numerous biotechnological applications. Plants uniquely synthesize an array of thioredoxins targeted to different cell compartments, for example chloroplastic f- and m-type thioredoxins involved in regulation of the Calvin-Benson cycle. The cytosolic h-type thioredoxins act as key regulators of seed germination and are recycled by NADPH-dependent thioredoxin reductase. The present review on thioredoxin h systems in plant seeds focuses on occurrence, reaction mechanisms, specificity, target protein identification, three-dimensional structure and various applications. The aim is to provide a general background as well as an update covering the most recent findings. This article is part of a Special Issue entitled: Plant Proteomics — a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. © 2016 Elsevier B.V.
Minerva Biotecnologica (11204826) 28(4)pp. 219-223
BACKGROUND: The main purpose of this study was to investigate the effect of an amino acid with side chain containing hydroxyl group in the active site on the activity of thioredoxins (Trxs). METHODS: A mutant protein with a threonine (Thr) substitution for the largely conserved glycine (Gly) at position 41 in the active site (WCGPC) of one of rice cytoplasmic Trx isoforms, OsTrx23, generated using site-directed mutagenesis and the activities of wild type (Wt) and mutant was compared using insulin assay under different pH. RESULT S: Insulin assay showed that whereas the activity of WtOsTrx23 were stable at different pH the activity of mutant G41TOsTrx23 was changed under different pH. CONCLUSIONS: The results indicate that the presence of Gly after second cysteine residue in the active site has an important role in stability of Trx under different pH conditions. The pH stability decreases considerably in the presence of a hydroxyl group in this position. © 2014 EDIZIONI MINERVA MEDICA.
Behnamfar, M.T. ,
Hadadzadeh h., H. ,
Simpson, J. ,
Darabi, F. ,
Shah piri, A. ,
Khayamian, T. ,
Ebrahimi, M. ,
Amiri rudbari, H. ,
Salimi, M. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy (13861425) 134pp. 502-516
Two mononuclear iron complexes, [Fe(tppz)2](PF6) 2·H2O (1) and Fe(tppz)Cl3· 2CHCl3 (2) where tppz is (2,3,5,6-tetra(2-pyridyl)pyrazine), have been synthesized and characterized by elemental analysis, spectroscopic methods (UV-Vis and IR) and single crystal X-ray structure analysis. The interaction of (1) as the nitrate salt ([Fe(tppz)2](NO3)2) with calf-thymus DNA (CT-DNA) has been monitored by UV-Vis spectroscopy, competitive fluorescence titration, circular dichroism (CD), voltammetric techniques, viscosity measurement, and gel electrophoresis. Gel electrophoresis of DNA with [Fe(tppz)2](NO3)2 demonstrated that the complex also has the ability to cleave supercoiled plasmid DNA. The results have indicated that the complex binds to CT-DNA by three binding modes, viz., electrostatic, groove and partial insertion of the pyridyl rings between the base stacks of double-stranded DNA. Molecular docking of [Fe(tppz) 2](NO3)2 with the DNA sequence d(ACCGACGTCGGT)2 suggests the complex fits into the major groove. The water-insoluble complex (2) can catalyze the cleavage of BSA at 40 °C. There are no reports of the catalytic effect of polypyridyl metal complexes on the BSA cleavage. Molecular docking of (2) with BSA suggests that, when the chloro ligands in the axial positions are replaced by water molecules, the BSA can interact with the Fe(III) complex more easily. © 2014 Elsevier B.V. All rights reserved.
International Journal of Biological Macromolecules (01418130) 73(1)pp. 154-159
Metallothioneins (MTs) are low-molecular weight proteins with high Cys content and a high affinity for metals. Plant MTs are classified into four types based on the arrangement of Cys in their amino acid sequences. In the present study, the gene encoding OsMTI-3a, a type 3 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into the Escherichia coli strain Rosetta (DE3). Following the induction with isopropyl β-d-1-thiogalactopyranoside, the OsMTI-3a was expressed as glutathione-S-transferase (GST)-tagged fusion protein. In comparison to control strain, the cells expressing GST-OsMTI-3a accumulated more Cd2+, Ni2+ and Zn2+ when they were grown in the medium containing CdCl2, NiCl2 or ZnSO4. The recombinant GST-OsMTI-3a was purified using affinity chromatography. The UV absorption spectra recorded after the reconstitution of the apo-protein with different metals confirmed that GST-OsMTI-3a was able to form complexes with Cd2+, Ni2+, and Zn2+. The reaction of the protein-metal complexes with 5-5-dithiobis (2-nitrobenzoic) revealed that the order of affinity of GST-OsMTI-3a toward different metals was Ni2+≥Cd2+>Zn2+>Cu2+. © 2014 Elsevier B.V..
Journal of the Science of Food and Agriculture (10970010) 95(1)pp. 141-147
BACKGROUND: Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the aleurone layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley aleurone layers is presented for the important enzymes in starch degradation: α-amylase and limit dextrinase (LD). RESULTS: Proteins were visualized by immunoblotting in aleurone layers and culture supernatants from dissected aleurone layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from aleurone layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from aleurone layers. CONCLUSIONS: Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated aleurone layers or aleurone layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from aleurone layers treated with GA+ABA or GA+SA. © 2014 Society of Chemical Industry.
International Journal of Biological Macromolecules (01418130) 74pp. 243-248
Rice (Oryza sativa L.) has multiple potential genes encoding thioredoxin (Trx) h and NADP-thioredoxin reductase (NTR). These NTR and Trx h isoforms, known as cytoplasmic NTR/Trx system along with multiple members of glutaredoxin (Grx) family constitute a complex redox control system in rice. In the present study, we investigated the kinetic parameters of two rice NTRs, OsNTRA and OsNTRB, toward three endogenous Trx h isoforms, OsTrx1, OsTrx20, and OsTrx23. The results showed that in contrast with OsTrx1 and OsTrx23, the isoform OsTrx20 was not reduced by OsNTR isoforms. The kcat/Km values of OsNTRB and OsNTRA toward OsTrx1 was six- and 13-fold higher than those values toward OsTrx23, respectively, suggesting that OsNTR isoforms do not reduce different OsTrx h isoforms, equivalently. Furthermore, the possible reduction of OsTrx isoforms by the glutathione (GSH)/Grx system was investigated through the heterologous expression of a gene encoding OsGrx9, a bicysteinic CPYC Grx found in rice. Whereas OsTrx23 was not reduced by GSH, OsTrx20 and with less efficiently OsTrx1 were reduced by GSH or GSH/Grx. Therefore, it seems that OsTrx1 can be reduced either by OsNTR or GSH/Grx. These data for the first time provides an evidence for cross-talking between NTR/Trx and GSH/Grx systems in rice. © 2014 Elsevier B.V.
Kirkensgaard, K.G. ,
Hägglund, P. ,
Shah piri, A. ,
Finnie, C. ,
Henriksen, A. ,
Svensson, B. Proteins: Structure, Function and Bioinformatics (10970134) 82(4)pp. 607-619
The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer of reducing equivalents from NADPH to Trx via a tightly NTR-bound flavin. Here, interactions between NTR and Trx are predicted by molecular modelling of the barley NTR:Trx complex (HvNTR2:HvTrxh2) and probed by site directed mutagenesis. Enzyme kinetics analysis reveals mutants in a loop of the flavin adenine dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent on these two residues, suggesting a distinct mode for NTR:Trx recognition. Comparison between the HvNTR2:HvTrxh2 model and the crystal structure of the Escherichia coli NTR:Trx complex reveals major differences in interactions involving the FAD- and NADPH-binding domains as supported by our experiments. Overall, the findings suggest that NTR:Trx interactions in different biological systems are fine-tuned by multiple intermolecular contacts. © 2013 Wiley Periodicals, Inc.
Plant Systematics and Evolution (16156110) 300(10)pp. 2199-2204
Thioredoxins (Trxs) are small proteins containing two redox-active half-Cys residues with the sequence -Cys-X-X-Cys- that function as protein disulfide reductases in a variety of biological reactions. The cytoplasmic type Trxs (h-type) constitutes a particularly large sub-group in higher plants. OsTrx20 and OsTrx23 are two Trx h isoforms in rice which were heterologously expressed in E. coli previously and found to be partially dimer under non-reducing conditions. In the present study, we substituted two vicinal Cys (Cys3 and Cys4) in N-terminal of OsTrx20 as well as the single Cys (Cys11) in N-terminal of OsTrx23 with Ser and provided the recombinant forms of mutants C3SOsTrx20, C4SOsTrx20, C3S-C4SOsTrx20 and C11SOsTrx23. The mutants C11SOsTrx23 and C3S-C4SOsTrx20 appeared exclusively in monomeric form under non-reducing conditions, whereas the mutants C3SOsTrx20 and C4SOsTrx20 were appeared in both monomeric and dimeric forms. These results suggest that Cys11 in OsTrx23 and both Cys3 and Cys 4 in OsTrx20 are involved in dimer formation. DTT-dependent insulin assay shows that the N-terminal Cys residues in OsTrx23 and OsTrx20 are not essential for protein activity. By contrast, the replacement of Cys57 with Ser which is located in active site of OsTrx20 shows that Cys 57 is not involved in dimerization, but has a critical role in reduction activity. © 2014, Springer-Verlag Wien.
International Journal of Biological Macromolecules (01418130) 68pp. 185-188
Thioredoxins are small (12-13kDa) ubiquitous proteins containing a redox active disulfide bridge. The primary structure of one of the rice Trx isoforms, OsTrx20, in which Thr is substituted for the largely conserved Gly in position 55 in the active site and Lys is substituted for the conserved Asp/Asn in position 48 is considerably different with other h-type Trx isoforms. In order to probe the functional roles of Thr-55 and Lys-48 in OsTrx20, Thr was replaced with Gly and Lys with Asp using site-directed mutagenesis. The wild type OsTrx20 as well as single mutants T55GOsTrx20, K48DOsTrx20 and the double mutant T55G-K48DOstrx20 were heterologously expressed in Escherichia coli and purified. The changes in the ability to reduce insulin for OsTrx20 and mutants as well as OsTrx23 which has a Trx typical active site were monitored in the pH range 6.5-8. The results showed that whereas the activity of wild type OsTrx20 is dependent on pH and decreases remarkably at high pH values, the activities of mutants T55GOsTrx20, K48DOsTrx20, T55G-K48DOsTrx20 and wild type OsTrx23 slightly change under different pH conditions. These results support the significant involvement of residues Thr-55 and Lys-48 in instability of OsTrx20 activity under pH variations. © 2014 Elsevier B.V.
Biotechnology and Applied Biochemistry (14708744) 60(3)pp. 275-282
Metallothioneins (MTs) are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. Plants have several MT isoforms, which are classified into four types based on the arrangement of Cys residues. In this study, two rice (Oryza sativa) MT isoforms, OsMTI-1b and OsMTII-1a from type 1 and type 4, respectively, were heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). Transformed cells expressing GST-OsMTI-1b showed increased tolerance to Ni 2+, Cd2+, and Zn2+ and accumulated more metal ions compared with cells expressing GST alone. However, heterologous expression of GST-OsMTII-1a had no significant effects on metal tolerance or ion accumulation. The UV absorption spectra and competitive reactions of in vitro Cd-incubated proteins with 5-5′-dithiobis(2-nitrobenzoic) acid revealed that GST-OsMTI-1b, but not GST-OsMTII-1a, is able to form Cd-thiolate clusters. Furthermore, heterologous expression of both GST-OsMTI-1b and GST-OsMTII-1a conferred H2O2 tolerance to E. coli cells. Taken together, the results presented here show that two different rice MT isoforms belonging to type 1 and type 4 differ in Ni2+, Cd2+, and Zn 2+ binding abilities, but they may have overlapping function in protection of cells against oxidative stress. © 2013 International Union of Biochemistry and Molecular Biology, Inc.
Protein Journal (18758355) 32(2)pp. 131-137
Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni2+, Cd2+, Zn2+ and Cu 2+ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni2+, Cd2+ and Zn2+ ions in vitro, whereas it has no substantial ability to bind Cu2+ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni2+/Cd2+ > Zn2+ > Cu2+ © 2013 Springer Science+Business Media New York.
Hägglund, P. ,
Björnberg, O. ,
Navrot, N. ,
Mørch jensen, J. ,
Maeda, K. ,
Kirkensgaard, K.G. ,
Shah piri, A. ,
Sultan, A. ,
Bunkenborg, J. ,
Gubler, F. Frontiers in Plant Science (1664462X) 4(MAY)
Thioredoxin (Trx) reduces disulfide bonds and play numerous important functions in plants. In cereal seeds, cytosolic h-type Trx facilitates the release of energy reserves during the germination process and is recycled by NADPH-dependent Trx reductase. This review presents a summary of the research conducted during the last 10 years to elucidate the structure and function of the barley seed Trx system at the molecular level combined with proteomic approaches to identify target proteins. © 2013 Hägglund, Björnberg, Navrot, Mørch Jensen, Maeda, Kirkensgaard, Shahpiri, Sultan, Bunkenborg, Gubler, Barrero, Henriksen, Finnie and Svensson.
Plant OMICS (18360661) 5(3)pp. 238-243
Thioredoxin h (Trx h) is a major cytoplamic and mitochondrial disulfide reductase. In plants, Trx h isoforms are encoded by a multigenic family of genes. The multiplicity of these isoforms raises the question of their functional specificity. In this study, we describe isolation and cloning of three cDNAs encoding different Trx h isoforms, namely OsTrx1, OsTrx20 and OsTrx23. Three Trx h were heterologously expressed in Escherichia coli and their activities were compared using DTT-dependent insulin assay. OsTrx23 and OsTrx1 demonstrated highest (0.05 Δ650/min) and lowest (0.016 Δ650/min) activity, respectively. In contrast to OsTrx1 and OsTrx20 isoforms, OsTrx23 was efficiently reduced by NADPH-dependent thioredoxin reductase from barley (HvNTR2). The gene expression of three Trx h was analyzed in one, two and three-week old rice seedlings. The transcripts of OsTrx23 with high intensity and OsTrx20 with low intensity were expressed in both root and shoot, whereas OsTrx1 was only expressed in root. All of three isoforms were appeared partially dimerized under non-reducing conditions suggesting that disulfide bridges were responsible for dimerization.
Protein Journal (18758355) 31(7)pp. 580-584
Infectious bursal disease is one of the most important viral diseases in the young chickens. VP2 protein is the major host protective immunogen of the virus. A hypervariable region is present in VP2 protein (hvVP2) that contains immunodominant epitops. The high hydrophobicity of hvVP2 region causes protein aggregation in Escherichia coli (E. coli). The objective of the present study was to improve the expression and the solubility of the hvVP2 protein in E. coli. The effects of fusion partners on the solubility of hvVP2 protein were studied. The protein was expressed in forms of unfused and N-terminally fused to GST and NusA. The results showed that the unfused hvVP2 protein was expressed in very low level. But, N-terminally fused hvVP2 protein to GST (glutathione-S-transferase) and NusA (N utilization substance A) showed significantly enhanced protein expression. The fusion of GST and hvVP2 was produced in aggregated form while in the presence of NusA, the hvVP2 protein was expressed in a soluble form. The NusA-hvVP2 protein was detected by a neutralizing monoclonal antibody, 1A6, in antigen-capture ELISA. In conclusion, the NusA protein is a suitable fusion partner to improve expression and solubility of the hvVP2 protein in E. coli. © 2012 Springer Science+Business Media, LLC.
Australian Journal of Crop Science (18352707) 6(6)pp. 1045-1050
An NADP/thioredoxin system, consisting of NADPH, NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx) plays a post-translational regulatory role by reducing disulfide bonds in target proteins involved in various cellular processes. Plants have a complex NTR/Trx system comprising several Trxs and NTR isoforms. Three genes encoding NTR were found in the genome of rice. OsNTRC is chloroplastic type NTR whereas OsNTRA and OsNTRB are cytoplasmic/mitochondrial type NTR. The presence of two isoforms of cytoplasmic/mitochondrial type NTR with high amino acid identity raises the question of their functional specificity. In the present study, we describe isolation and cloning of two cDNAs encoding OsNTRA and OsNTRB. Both isoforms were heterologously expressed in Escherichia coli and purified using affinity chromatography, enabling comparison of their NADPHdependent reduction activities. OsNTRB and OsNTRA exhibited almost similar in vitro activities. OsNTRB was highly expressed in roots and shoots of one, two and three week-old rice seedlings, whereas the expression of OsNTRA in shoots was much higher than roots. Differential expression of OsNTRA in different tissues suggests that this isoform may have different functions in different tissues.
Proteomics (16159861) 11(9)pp. 1595-1605
The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Phytochemistry (00319422) 71(14-15)pp. 1650-1656
Enzymes involved in redox control are important during seed germination and seedling growth. Ascorbate-glutathione cycle enzymes in barley embryo extracts were monitored both by 2D-gel electrophoresis and activity measurements from 4 to 144 h post imbibition (PI). Strikingly different activity profiles were observed. No ascorbate peroxidase (APX) activity was present in mature seeds but activity was detected after 24 h PI and increased 14-fold up to 144 h PI. In contrast, dehydroascorbate reductase (DHAR) activity was present at 4 h PI and first decreased by 9-fold until 72 h PI followed by a 5-fold increase at 144 h PI. Glutathione reductase and monodehydroascorbate reductase activities were also detected at 4 h PI, and showed modest increases of 1.8- and 2.7-fold, respectively, by 144 h PI. The combination of functional analysis with the proteomics approach enabled correlation of the activity profiles and protein abundance. While gel spots containing APX showed intensity changes consistent with the activity profile from 0 to 72 h PI, DHAR spot intensities indicated that post-translational regulation may be responsible for the observed changes in activity. Transcript profiling, 2D-western blotting and mass spectrometric characterization of multiple APX spots demonstrated the presence of APX1 and minor amounts of APX2. © 2010 Elsevier Ltd. All rights reserved.
Molecular Plant (16742052) 2(3)pp. 378-389
Thioredoxins (Trx) are ubiquitous proteins that participate in thiol disulfide reactions via two active site cysteine residues, allowing Trx to reduce disulfide bonds in target proteins. Recent progress in proteome analysis has resulted in identification of a wide range of potential target proteins for Trx, indicating that Trx plays a key role in several aspects of cell metabolism. In contrast to other organisms, plants contain multiple forms of Trx that are classified based on their primary structures and sub-cellular localization. The reduction of cytosolic and mitochondrial types of Trx is dependent on NADPH and catalyzed by NADPH-dependent thioredoxin reductase (NTR). In barley, two isoforms each of Trx and NTR have been identified and investigated using proteomics, gene expression, and structural studies. This review outlines the diverse roles suggested for cytosolic/mitochondrial-type Trx systems in cereal seeds and summarizes the current knowledge of the barley system including recent data on function, regulation, interactions, and structure. Directions for future research are discussed. © The Author 2009.
Plant Physiology (00320889) 146(2)pp. 789-799
The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer. © 2007 American Society of Plant Biologists.
