Arabidopsis thaliana NAPHP Thioredoxin Reductase

Jacquot, Jean‐Pierre; Rivera‐Madrid, Renata; Marinho, Paulo; Kollárová, Marta; Maréchal, Pierre Le; Miginiac‐Maslow, Myroslawa; Meyer, Yves

doi:10.1006/jmbi.1994.1091

Cited by 127 publications

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“…Plasmid pET-Ch 1 [25] was used as pH 6.0. Recombinant NTR from Arabidopsis was purified as dea template with the following oligonucleotides (mutated bases scribed in Jacquot et al [33]. Purification of recombinant WT in bold characters) :…”

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confidence: 99%

The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 ¹H‐¹³C NMR

Krimm

Lemaire

Ruelland

et al. 1998

European Journal of Biochemistry

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The role of the invariant Trp residue at the redox site of thioredoxins was investigated by sitedirected mutagenesis of a Chlamydomonas reinhardtii thioredoxin h. Though being still redox active with NADPHϪthioredoxin reductase and chemical substrates [dithiothreitol and 5,5′-dithio-bis(2-nitrobenzoic acid)] the Trp35→Ala-mutated protein completely lost the capacity to activate the thiol-regulated NADPH-dependent malate dehydrogenase. However, it was able to activate a mutant malate dehydrogenase where only the most exposed disulfide was retained. The pH dependence of the redox-site Cysβ 1 H/ 13 C-NMR frequencies of the wild-type and mutated proteins, in both the reduced and oxidised states, were compared over the pH range 5.8Ϫ10. The mutation does not affect the conserved buried Asp30, which titrates with a pK a of 7.5 in the oxidised proteins in agreement with previous studies. However, for the reduced forms of the proteins, the pH dependence of resonances of both Cys was strongly affected by the mutation. In the case of the wild-type thioredoxin, two apparent pK a values were found around 7.0 and 9.5 and could be assigned to the titration of Cys36 and Cys39 thiol, respectively, similar to the case of Escherichia coli thioredoxin. For the mutated thioredoxin a single pK a was found around 8.3. This result can be interpreted as a single pK a of either Cys36 or Cys39 or both. While the mutation clearly affects ionisations, the measured redox potentials of the active-site Cys pair are not significantly affected by the Trp35→Ala mutation. Possible roles of an aromatic side chain on the reactivity of the catalytic Cys residues in thioredoxins are proposed.Keywords : thioredoxin h; Chlamydomonas reinhardtii; redox potential ; NMR ; cysteine pK a.Thioredoxins are small globular proteins of 100Ϫ120 amino undergo a two-electron oxidation, with a redox potential of acids present in all living cells and represent, with the bovine about Ϫ270 mV, to form a disulfide [9]. The active Cys residues pancreatic trypsin inhibitor (BPTI), one of the classes of proteins are included in a geometrically conserved environment organbest characterised in terms of structure either by radio-ised around a protruding first turn of helix composed of a invaricrystallography [1Ϫ3] Trp side chain is involved in a hydrogen bond between N 1 H and nase ; Nbs2, 5,5′-dithio-bis(2-nitrobenzoic acid) ; NTR, NADPH-thiore-the carboxylate of an invariant exposed Asp residue (Asp61 in doxin reductase; TOCSY, total correlation spectroscopy ; WT, wild type; E. coli thioredoxin). Overall, such a structural feature resembles mBBr, monobromobimane.

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The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 ¹H‐¹³C NMR

Krimm

Lemaire

Ruelland

et al. 1998

European Journal of Biochemistry

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“…coli thioredoxin reductase has been cloned and sequenced [21,22] and its biochemical and physical properties extensively studied [23,24]. Eukaryotic thioredoxin reductases have so far been only cloned from Penicillium chrysogenum [25], Saccharomyces cerevisiae [26], and Arabidopsis thaliana [27] and they show 44-50% sequence identity to the bacterial enzyme. We now report the cloning and sequencing of a putative thioredoxin reductase from human placenta.…”

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Cloning and sequencing of a human thioredoxin reductase

et al. 1995

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E. coli thioredoxin reductase has been cloned and sequenced [21,22] and its biochemical and physical properties extensively studied [23,24]. Eukaryotic thioredoxin reductases have so far been only cloned from Penicillium chrysogenum [25], Saccharomyces cerevisiae [26], and Arabidopsis thaliana [27] and they show 44-50% sequence identity to the bacterial enzyme. We now report the cloning and sequencing of a putative thioredoxin reductase from human placenta.

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“…The Trx assay using 5,5?-Dithio-bis-(2-nitrobenzoic acid) (DTNB) was carried out as described (Jacquot et al 1994). The NTR activity (Jacquot et al 1994) was measured spectrophotometrically.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…The NTR activity (Jacquot et al 1994) was measured spectrophotometrically. One unit of enzyme was defined as the amount necessary to decompose 1 mmol of substrate per min at 258C.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…Trx assay (Jacquot et al 1978(Jacquot et al , 1994 was performed after an incubation period of 30, 120, and 240 min at 48C. The formation of Trx h3 and f dimers was analyzed by SDS-PAGE after incubation of proteins with Cd ions in the absence or in the presence of 30 mM b-mercaptoethanol (b-MET) and dithiothreitol (DTT).…”

Section: Effect Of CD Ions On Trx In Vitromentioning

confidence: 99%

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Cadmium affects the NADP-thioredoxin reductase/thioredoxin system in germinating pea seeds

Smiri

Jelali

Ghoul

2013

Journal of Plant Interactions

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Seeds have devised sophisticated mechanisms to cope with adverse situations. Among these mechanisms the collectively referred to as redox homeostasis. We have investigated the thioredoxin (Trx)/NADPH/NADPHdependent thioredoxin reductase (NTR) systems during the pea (Pisum sativum L.) seed germination under heavy metal stress. Cotyledon and embryonic axis were analyzed between 0 and 5 days of germination in the presence or absence of cadmium (Cd). Under Cd stress conditions, NTR activity and Trx h3 and Trx h4 expression were stimulated, while the overall activity of Trx decreased. When incubated with Cd ions in vitro, the disulfide reductase activity of Trx h3 and f isoforms was drastically inhibited, probably through the formation of protein dimers as evidenced by the electrophoresis analysis. The NADPH status was also affected, since Cd treatment provoked an increase in oxidized state of coenzyme as compared to control redox ratio. The contribution of Trx system to the redox regulation during the germination is discussed in relation with the Cd-caused oxidative stress.

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Arabidopsis thaliana NAPHP Thioredoxin Reductase

Cited by 127 publications

References 0 publications

The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 ¹H‐¹³C NMR

The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 ¹H‐¹³C NMR

Cloning and sequencing of a human thioredoxin reductase

Cadmium affects the NADP-thioredoxin reductase/thioredoxin system in germinating pea seeds

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Arabidopsis thaliana NAPHP Thioredoxin Reductase

Cited by 127 publications

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The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 1H‐13C NMR

The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 1H‐13C NMR

Cloning and sequencing of a human thioredoxin reductase

Cadmium affects the NADP-thioredoxin reductase/thioredoxin system in germinating pea seeds

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The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 ¹H‐¹³C NMR

The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 ¹H‐¹³C NMR