Cysteine‐153 is required for redox regulation of pea chloroplast fructose‐1,6‐bisphosphatase

Jacquot, Jean‐Pierre; Lopéz-Jaramillo, Javier; Miginiac‐Maslow, Myroslawa; Lemaire, Stéphane D.; Cherfils, Jacqueline; Chueca, Ana; López-Gorgé, J

doi:10.1016/s0014-5793(96)01459-7

Cited by 101 publications

(63 citation statements)

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“…conditions (this was also observed for fructose-1,6-bisphosphatase and ferredoxin [30,31]). It is believed that in a complex We have investigated a Trp→Ala site-directed thioredoxin h mutant of Chlamydomonas reinhardtii [25,26,7].…”

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confidence: 59%

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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confidence: 59%

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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“…We would have expected the same effect after exchange of Cys-428 for Ser; however, autoinhibition was only reduced but not completely abolished in this particular mutant. Similar observations were made with GAPDH (Sparla et al, 2002) and Fru-1,6-bisP phosphatase (Jacquot et al, 1997), where the exchange of only one of the two Cys residues involved in regulatory disulfide formation did not result in a complete loss of redox dependency. Simultaneous exchange of both Cys-420 and Cys-428 also completely abolished autoinhibition.…”

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confidence: 80%

An Autoinhibitory Domain Confers Redox Regulation to Maize Glycerate Kinase

Bartsch

Mikkat²,

Hagemann

et al. 2010

Plant Physiology

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Glycerate 3-kinase (GLYK) is the terminal enzyme of the photorespiratory cycle in plants and many cyanobacteria. For several C 4 plants, notably grasses of the NADP-malic enzyme (ME) subtype, redox regulation of GLYK has been reported, but the responsible molecular mechanism is not known. We have analyzed the enzyme from the NADP-ME C 4 plant maize (Zea mays) and found that maize GLYK, in contrast to the enzyme from C 3 plants and a dicotyledonous NADP-ME C 4 plant, harbors a short carboxy-terminal extension. In its oxidized (night) form, a disulfide bridge is formed between the two cysteine residues present in this extra domain, and GLYK activity becomes inhibited. Cleavage of this bond by thioredoxin f produces the fully active thiol form, releasing autoinhibition. Fusion of the maize GLYK redox-regulatory domain to GLYK from C 3 plants confers redox regulation to these otherwise unregulated enzymes. It appears that redox regulation of GLYK could be an exclusive feature of monocotyledonous C 4 plants of the NADP-ME type, in which linear electron transport occurs only in the mesophyll chloroplasts. Hence, we suggest that GLYK, in addition to its function in photorespiration, provides glycerate 3-phosphate for the accelerated production of triose phosphate and its export from the mesophyll. This could facilitate the activation of redoxregulated Calvin cycle enzymes and the buildup of Calvin cycle intermediates in the bundle sheath of these particular C 4 plants during the dark/light transition.

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“…24 and this study) suggests a disulfide bond between Cys 20 and Cys 289 in the A subunit and another one between the corresponding Cys residues in the B subunit. Similarly in both malate dehydrogenase (24,25) and fructose bisphosphatase (26), at least one additional disulfide bond not seen in the available crystal structures seems possible. Homology modeling, based on the coordinates of the NAD-linked B. stearothermophilus enzyme in PDB code 1gd1 (12), implicates the Cys residues corresponding to Cys 20 and Cys 289 in the dark inactivation of the NADP-linked Chlamydomonas reinhardtii enzyme.…”

Section: Discussionmentioning

confidence: 98%

Chloroplast Glyceraldehyde-3-phosphate Dehydrogenase Contains a Single Disulfide Bond Located in the C-terminal Extension to the B Subunit

Isupov

Littlechild

et al. 2001

Journal of Biological Chemistry

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Mass mapping analysis based on cyanylation and CNinduced cleavage indicates that the two cysteine residues in the C-terminal extension of the B subunit of the light-activated pea leaf chloroplast glyceraldehyde-3-phosphate dehydrogenase form a disulfide bond. No evidence was found for a disulfide bond in the A subunit, nor was there any indication of a second disulfide bond in the B subunit. The availability of the structure of the extended glyceraldehyde-3-phosphate dehydrogenase from the archaeon Sulfolobus solfataricus allows modeling of the B subunit. As modeled, the two cysteine residues in the extension are positioned to form an interdomain disulfide cross-link.The NADP-linked chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) 1 (EC 1.2.1.13) of flowering plants consist of two subunits. The A subunit is similar in length and in sequence to the eucaryotic and eubacterial NAD-linked enzyme (EC 1.2.1.12) protomers. The B subunit has a 27-to 29-residue C-terminal extension that contains two invariant cysteines (Fig. 1). B subunits have only been found in angiosperms. In species ranging from Cyanobacteria to angiosperms, NADP-linked glyceraldehyde-3-phosphate dehydrogenases are light-activated (1, 2). Activation involves the reduction of an inhibiting disulfide bond or bonds. Although modeling indicates that two Cys residues in the A subunit are almost certainly responsible for the redox sensitivity of the enzyme in green algae and nonflowering plants (3), the presence of the conserved Cys pair in the extended angiosperm B subunit suggests that it too might be involved in redox regulation. The purpose of the present experiments was to determine which cysteine residues in the pea chloroplast glyceraldehyde-3-phosphate dehydrogenase form cystines and are responsible for the redox sensitivity of the enzyme. MATERIALS AND METHODSEnzyme Isolation-The wild type NADP-linked glyceraldehyde-3-phosphate dehydrogenase used in the mass mapping experiments was purified from pea plants by a modification of the method of Anderson et al. (4). Prior to the acetone fractionation step, the suspended MgCl 2 -polyethylene glycol fraction was passed through a DEAE column (10 ml of Sigma Fast Flow DEAE) in 10 mM KHPO 4 , pH 7.8 buffer. The glyceraldehyde-3-phosphate dehydrogenase in this fraction was not retained on the column. It emerged in a milky excluded fraction. After acetone fractionation, the cloudy 50% acetone supernatant was applied to a second 10-ml DEAE column in 10 mM Tris-HCl, pH 7.8, and the column was washed overnight with the Tris buffer and eluted with a linear phosphate gradient (200 ml of 10 mM KHPO 4 , pH 7.8 and 200 ml of 0.4 M KHPO 4 , pH 7.0). In some experiments, including those represented by Figs. 2 and 3, 10 mM mercaptoethanol was included in the buffers. In other experiments, including the experiments represented by Figs. 4 and 5, mercaptoethanol was omitted. Essentially identical results were obtained with or without inclusion of mercaptoethanol in the buffers during isolation. Chromatograph...

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Cysteine‐153 is required for redox regulation of pea chloroplast fructose‐1,6‐bisphosphatase

Cited by 101 publications

References 29 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

An Autoinhibitory Domain Confers Redox Regulation to Maize Glycerate Kinase

Chloroplast Glyceraldehyde-3-phosphate Dehydrogenase Contains a Single Disulfide Bond Located in the C-terminal Extension to the B Subunit

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Cysteine‐153 is required for redox regulation of pea chloroplast fructose‐1,6‐bisphosphatase

Cited by 101 publications

References 29 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

An Autoinhibitory Domain Confers Redox Regulation to Maize Glycerate Kinase

Chloroplast Glyceraldehyde-3-phosphate Dehydrogenase Contains a Single Disulfide Bond Located in the C-terminal Extension to the B Subunit

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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