Redox signalling in the chloroplast: structure of oxidized pea fructose-1,6-bisphosphate phosphatase

Chiadmi, M.

doi:10.1093/emboj/18.23.6809

Cited by 134 publications

(116 citation statements)

References 47 publications

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“…Consistent with the present results, when the C-terminal extension is removed genetically, the resultant recombinant mutant enzyme is significantly less redox-sensitive (5,18). The regulatory disulfide bonds in the redox-regulated chloroplast malate dehydrogenase (20,21) and in the chloroplast fructose bisphosphatase (22) are also located in extensions or insertions. Modeling suggests that the two Cys residues in the C-terminal extension of the chloroplast glyceraldehyde-3-phosphate dehydrogenase are located in different domains.…”

Section: Discussionsupporting

confidence: 90%

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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Section: Discussionsupporting

confidence: 90%

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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“…FBPase from the chloroplast evidently does not bind AMP (30,31). Yet existing crystal structures of reduced and oxidized chloroplast FBPase have nearly the same quaternary arrangement of subunits, resembling most closely the T-state of mammalian FBPase.…”

Section: Expression Purification and Secondary Structure Analysis Omentioning

confidence: 98%

“…Interestingly, position 18 of the chloroplast enzyme, which corresponds to position 10 of mammalian FBPase, is isoleucine, this residue being widely conserved among FBPases. Furthermore, in the inactive, oxidized form of chloroplast FBPase, loop 61-81 (corresponding to loop 52-72 of the mammalian enzyme) is in the disengaged conformation (30), whereas the equivalent loop in the active, reduced form of chloroplast FBPase is in the disordered conformation (32). Evidently, oxidation and reduction of the disulfide bond in chloroplast FBPase influences the relative stability of the disengaged loop-conformation in a manner similar to that of AMP in mammalian FBPases.…”

Section: Expression Purification and Secondary Structure Analysis Omentioning

confidence: 98%

“…The thioredoxin-mediated formation of a disulfide bond between Cys 153 and Cys 173 in chloroplast FBPase (29) putatively stabilizes the position of a loop that excludes the binding of metal activators and the engaged conformation of loop 61-81 (which corresponds to loop 52-72 of mammalian FBPase) (30). FBPase from the chloroplast evidently does not bind AMP (30,31).…”

Section: Expression Purification and Secondary Structure Analysis Omentioning

confidence: 99%

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The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Nelson

Kurbanov

Honzatko

et al. 2001

Journal of Biological Chemistry

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Residues 1-10 of porcine fructose-1,6-bisphosphatase (FBPase) are poorly ordered or are in different conformations, sensitive to the state of ligation of the enzyme. Deletion of the first 10 residues of FBPase reduces k cat by 30-fold and Mg 2؉ affinity by 20-fold and eliminates cooperativity in Mg 2؉ activation. Although a fluorescent analogue of AMP binds with high affinity to the truncated enzyme, AMP itself potently inhibits only 50% of the enzyme activity. Additional inhibition occurs only when the concentration of AMP exceeds 10 mM. Deletion of the first seven residues reduces k cat and Mg 2؉ affinity significantly but has no effect on AMP inhibition. The mutation of Asp 9 to alanine reproduces the weakened affinity for Mg 2؉ observed in the deletion mutants, and the mutation of Ile 10 to aspartate reproduces the AMP inhibition of the 10-residue deletion mutant. Changes in the relative stability of the known conformational states for loop 52-72, in response to changes in the quaternary structure of FBPase, can account for the phenomena above. Some aspects of the proposed model may be relevant to all forms of FBPase, including the thioredoxinregulated FBPase from the chloroplast.Fructose-1,6-bisphosphatase (D-fructose 1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase 1 ) catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate and inorganic phosphate (P i ) (1-3). The reaction facilitated by FBPase is subject to hormone and metabolite regulation, the net result of which is the tight coordination of FBPase and fructose 6-phosphate 1-kinase activities (4). FBPase activity requires divalent cations such as Mg 2ϩ , Mn 2ϩ , or Zn 2ϩ , and plots of velocity versus metal ion concentration are sigmoidal with a Hill coefficient of 2.0 (5-7). AMP binds cooperatively (Hill coefficient of 2) 28 Å from the nearest active site (8) and inhibits the enzyme, whereas F26P 2 binds at the active site (9). Inhibition of FBPase by AMP and F26P 2 is synergistic. F26P 2 can lower the apparent inhibition constant for AMP by up to 10-fold (6).FBPase is a tetramer of identical subunits (M r ϭ 37,000). To a first approximation, these subunits lie in the same plane and occupy the corners of a square in one of the principal quaternary states of the mammalian enzyme (R-state). By past convention, subunit C1 occupies the upper left-hand corner, with subunits C2-C4 following in a clockwise direction. AMP causes a transition from the R-state to the T-state, driving a 17°r otation of the C1-C2 subunit pair with respect to the C3-C4 pair about a molecular 2-fold axis of symmetry (10). Complexes of FBPase with AMP in the presence of F16P 2 , F26P 2 , and fructose 6-phosphate are all in the T-state (9, 11, 12), whereas in the absence of AMP, the enzyme has appeared in the R-state in crystal structures (13,14).Mutations in loop 52-72 and in the hinge preceding the loop (residues 50 -51) greatly influence catalysis and AMP inhibition of FBPase and together suggest the necessity of an engaged conformation for ...

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“…It is known that both enzymes are redoxregulated but the relationship between them is still unclear at the molecular level. Although we know precisely the structure and regulatory properties of pea FBPase, little is known about the structure-function relationship of SBPase (Jacquot et al 1995;Chiadmi et al 1999). In a first series of experiments, we have produced the corresponding recombinant proteins from poplar and Chlamydomonas reinhardtii but the enzymes turned out not to be sufficiently stable and rather rapidly denatured.…”

Section: Biochemical and Structural Studies Of Redox Enzymes In Progressmentioning

confidence: 99%

Structural and functional characterization of tree proteins involved in redox regulation: a new frontier in forest science

Jacquot

Couturier

Didierjean

et al. 2016

Annals of Forest Science

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& Key message This paper describes how the combination of genomics, genetic engineering, and 3D structural characterization has helped clarify the redox regulatory networks in poplar with consequences not only in system biology in plants but also in bacteria and mammalian systems. & Context Tree genomes are increasingly available with a large number of orphan genes coding for proteins, the function of which is still unknown.& Aims and methods Modern techniques of genome analysis coupled with recombinant protein technology and massive 3D structural determination of tree proteins should help elucidate the function of many of the proteins encoded by orphan genes. X-ray crystallography and NMR will be the methods of choice for protein structure determination. & Results In this review, we provide examples illustrating how the above-mentioned techniques improved our understanding of redox regulatory circuits in poplar, the first forest tree species sequenced. We showed that poplar peroxiredoxins use either thioredoxin or glutaredoxin as electron donors toHandling Editor: Jean-Michel Leban Executive summary This review describes the successful use of technologies to overproduce and purify recombinant enzymes from trees with the aim of solving their 3D structures and identifying their molecular interactions either with other proteins or with potential substrates. Currently only ca a dozen of tree genomes have been annotated and released including six forest species, but this will change rapidly with the oncoming release of the chestnut and oak genomes notably. The availability of additional genomes will open the way to identifying the function of a large number of orphan gene products, one of the ways to characterize these functions being to produce and analyze the corresponding protein products. reduce hydrogen peroxide. That glutaredoxin could be a reductant was unknown at the time of this discovery even in other biological organisms and was later confirmed notably by the observation that the two genes are fused in some bacteria and by the resolution of the structure of the bacterial hybrid protein. Similarly, genome analysis coupled to in vitro analysis of enzymatic properties led to the discovery that some plant methionine sulfoxide reductases can also use both thioredoxins and glutaredoxins as electron donors. Besides their disulfide reductase activity, it has been demonstrated that some poplar glutaredoxins are also involved in iron-sulfur center biogenesis and assembly. The original 3D structure determination has been made with poplar glutaredoxin C1 and then confirmed in a variety of other biological organisms including human. Our work also showed that in plants, socalled glutathione peroxidases use thioredoxins and not glutathione as electron donors. This is true for all nonselenocysteine-containing glutathione peroxidases. Finally, connections between the thioredoxin and glutaredoxin systems have been elucidated through the study of atypical poplar thioredoxins. & Conclusion Altogether, these data il...

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Redox signalling in the chloroplast: structure of oxidized pea fructose-1,6-bisphosphate phosphatase

Cited by 134 publications

References 47 publications

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

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

The N-terminal Segment of Recombinant Porcine Fructose-1,6-bisphosphatase Participates in the Allosteric Regulation of Catalysis

Structural and functional characterization of tree proteins involved in redox regulation: a new frontier in forest science

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