Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii

Decottignies, Paulette; Schmitter, Jean‐Marie; Jacquot, Jean‐Pierre; Dutka, Sophie; Picaud, Alain; Gadal, Pierre

doi:10.1016/0003-9861(90)90525-4

Cited by 42 publications

(33 citation statements)

References 44 publications

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“…The activity was expressed as rate of reduction of thioredoxin, using 13600 M-' cm-' as the molar absorption coefficient of Nbsz (2 mol SH being formed/mol thioredoxin reduced) (Table 2) revealed no difference between the two bacterial proteins. In addition, the observed rates were comparable to that obtained with thioredoxin m from spinach chloroplasts [24]. On the other hand, the activation of FBPase revealed differences between the thioredoxins.…”

Section: Thioredoxin Reductase ( N a D P H )supporting

confidence: 62%

“…At least one of the targets, namely FBPase, shows a very specific requirement for thioredoxinf [33]. NADP-MDH, while being activated preferentially by thioredoxin nz, can also be reduced by other thioredoxins [24]. Therefore, these two plant target enzymes appeared to be an appropriate test for a structure/ function relationship study on thioredoxin.…”

Section: Discussionmentioning

confidence: 99%

“…Considering that E. coli thioredoxin was a good substitute for spinach thioredoxin m [24], we modified it by site-directed mutagenesis in order to investigate which residues could be important for thioredoxin ,f;iFBPase interaction specificity. Asn61 seemed to be such a residue.…”

Section: Discussionmentioning

confidence: 99%

“…More than 20 thioredoxin primary structures are now available and all of them share the characteristic active site [7]. Surprisingly, the m-type spinach thioredoxin shows more than a 50% amino acid similarity with E. coli thioredoxin [23] and exhibits similar kinetic properties in NADP-MDH activation [24]. On the other hand, the primary structure of J-type spinach thioredoxin is very different from all other thioredoxins whose primary structures are known [25].…”

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confidence: 99%

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Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes

Lamotte

Miginiac‐Maslow

Decottignies

et al. 1991

European Journal of Biochemistry

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A new over-expression system has been set up for Escherichiu coli thioredoxin, yielding 55 mg purified protein/ 10 g fresh cells. This system has been used to produce thioredoxin modified by site-directed mutagenesis. Taking advantage of the structural and enzymatic similarity between E. coli and spinach m-type thioredoxin, Asp61 of E. coli thioredoxin has been changed into Asn in order to investigate the impact of the suppression of a charged residue on the interaction of thioredoxin with target enzymes. The modification did not significantly alter the structure of the protein. Neither the rate of reduction of insulin and 5,5'-dithio-bis(2-nitrobenzoic acid) by the reduced thioredoxin, nor the reduction by NADPH-dependent thioredoxin reductase, have been modified. The major effect of the mutation was observed for chloroplast enzyme activation with thioredoxin reduced by dithiothreitol and with thioredoxin reduced by ferredoxin-dependent thioredoxin reductase in a light-activation reconstituted chloroplast system. The substitution of the negatively charged Asp61 by the neutral Asn led to an increase in the efficiency of spinach fructose-l,6-bisphosphatase activation by the dithiothreitol-reduced thioredoxin, and to an increase in both spinach fructose-l,6-bisphosphatase and corn NADP-dependent malate dehydrogenase activities in the light-activation system. This suggests that the suppression of the negative charge improves the reactivity of thioredoxin with chloroplast enzymes such as fructose-l,6-bisphosphatase and ferredoxin-dependent thioredoxin reductase.

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Section: Thioredoxin Reductase ( N a D P H )supporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes

Lamotte

Miginiac‐Maslow

Decottignies

et al. 1991

European Journal of Biochemistry

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“…Except for spinach PRK (see below), the recombinant proteins were all expressed in E. coli. Spinach thioredoxins f and m and spinach FBPase (26) and spinach FTR (27) were purified as described previously, as were pea thioredoxin f (7), C. reinhardtii thioredoxin m (28), and pea FBPase (29,30). Spinach PRK was isolated from a transformed strain of the yeast Pichia pastoris that carries the gene for the wild-type enzyme (31) and was purified as described previously (24).…”

Section: Methodsmentioning

confidence: 99%

Oxidation-Reduction Properties of Chloroplast Thioredoxins, Ferredoxin:Thioredoxin Reductase, and Thioredoxin f-Regulated Enzymes

et al. 1999

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Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (E m ) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values g 7.0, but with two components being present at pH values <7.0. The slopes of plots of E m versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i.e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the E m versus pH profile for PRK shows three regions, consistent with the presence of pK a values for the two regulatory cysteines in the region between pH 7.5 and 9.0.The ferredoxin/thioredoxin system of oxygenic photosynthetic organisms plays an important role in the regulation of the carbon metabolism of these organisms (1-3). The initial step in the thioredoxin regulatory pathway, which has been extensively characterized in spinach and pea chloroplasts, involves the reduction of ferredoxin:thioredoxin reductase (hereafter abbreviated FTR 1 ) by the reduced ferredoxin generated during light-driven noncyclic electron flow (1-3). Spinach leaf FTR, the best characterized of these enzymes, is a 25.6 kDa heterodimeric protein located in the chloroplast stroma (1-3). FTR contains a unique cluster that serves to stabilize the one-electron-reduced intermediate formed after the first electron donation by ferredoxin, during the two-electron reduction of the activesite disulfide of the oxidized enzyme to the two cysteine thiols present in reduced FTR (4, 5). FTR reduces thioredoxin in a reaction in which the two cysteines at the active site of the reduced enzyme become oxidized to a cystine disulfide, while the active-site disulfide of the oxidized thioredoxin becomes reduced to two cysteine thiols (1-5). FTR reduces both of the thioredoxins found in chloroplasts, thioredoxin f and thioredoxin m (monomeric proteins with molecular masses of ∼12 kDa that contain a conserved -WCGPCactive site), with equal efficiency. However, the two chloroplast thioredoxins display differential but overlapping reactivities among the array of identified target proteins (1-3). Although regulatory reduction by thioredoxin m appears to be restricted to glucose-6-phosph...

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Ferredoxin and Ferredoxin-Dependent Enzymes

Knaff

1996

Oxygenic Photosynthesis: The Light Reactions

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Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii

Cited by 42 publications

References 44 publications

Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes

Mutation of a negatively charged amino acid in thioredoxin modifies its reactivity with chloroplastic enzymes

Oxidation-Reduction Properties of Chloroplast Thioredoxins, Ferredoxin:Thioredoxin Reductase, and Thioredoxin f-Regulated Enzymes

Ferredoxin and Ferredoxin-Dependent Enzymes

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