Catherine Corbier

Structure-function relationship of the chloroplastic glutaredoxin S12 with an atypical WCSYS active site.

Glutaredoxins (Grxs) are efficient catalysts for the reduction of mixed disulfides in glutathionylated proteins, using glutathione or thioredoxin reductases for their regeneration. Using GFP fusion, we have shown that poplar GrxS12, which possesses a monothiol 28WCSYS32 active site, is localized in chloroplasts. In the presence of reduced glutathione, the recombinant protein is able to reduce in vitro substrates, such as hydroxyethyldisulfide and dehydroascorbate, and to regenerate the glutathionylated glyceraldehyde-3-phosphate dehydrogenase. Although the protein possesses two conserved cysteines, it is functioning through a monothiol mechanism, the conserved C terminus cysteine (Cys87) being dispensable, since the C87S variant is fully active in all activity assays. Biochemical and crystallographic studies revealed that Cys87 exhibits a certain reactivity, since its pKa is around 5.6. Coupled with thiol titration, fluorescence, and mass spectrometry analyses, the resolution of poplar GrxS12 x-ray crystal structure shows that the only oxidation state is a glutathionylated derivative of the active site cysteine (Cys29) and that the enzyme does not form inter- or intramolecular disulfides. Contrary to some plant Grxs, GrxS12 does not incorporate an iron-sulfur cluster in its wild-type form, but when the active site is mutated into YCSYS, it binds a [2Fe-2S] cluster, indicating that the single Trp residue prevents this incorporation.

Thioredoxins and related proteins in photosynthetic organisms: molecular basis for thiol dependent regulation

Thioredoxins are small molecular weight disulfide oxidoreductases specialized in the reduction of disulfide bonds on other proteins. Generally, the enzymes which are selectively and reversibly reduced by these proteins oscillate between an oxidized and inactive conformation and a reduced and active conformation. Thioredoxin constitutes the archetype of a family of protein disulfide oxidoreductases which comprises glutaredoxin and protein disulfide isomerase. Thioredoxin and glutaredoxin serve many roles in the cell, including the redox regulation of target enzymes and transcription factors. They can also serve as hydrogen donors to peroxiredoxins, recently discovered heme free peroxidases, the function of which is to get rid of hydroperoxides in the cell. This review describes the molecular basis for the functioning and interaction between these enzymes in photosynthetic organisms.

An Atypical Catalytic Mechanism Involving Three Cysteines of Thioredoxin.

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys4) and two (Cys58 and Cys61) in the classical thioredoxin active site (57WCGPC61). The property of a mutant in which Cys58 was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys4 is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys58 can be formed, linking it to either Cys61 or Cys4. We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys4 to convert this atypical thioredoxin h back to its active reduced form.

Redox based anti-oxidant systems in plants: Biochemical and structural analyses

We provide in this paper a comparative biochemical and structural analysis of the major thiol oxidoreductases (thioredoxin and glutaredoxin) of photosynthetic organisms in relation with their reductases and with target proteins, especially those involved either in the detoxication of peroxides such as hydrogen peroxide (thiol-peroxidases) or in the repair of oxidized methionines in proteins (methionine sulfoxide reductases). Particular emphasis will be given to the catalytic and regeneration mechanisms used by these enzymes. In addition, the protein-protein interactions of these systems will be discussed, leading to an integrated view of the functioning of these systems in various plant sub-cellular compartments.

Trapping of the Thioacylglyceraldehyde-3-phosphate Dehydrogenase Intermediate from Bacillus stearothermophilus: DIRECT EVIDENCE FOR A FLIP-FLOP MECHANISM

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8Å resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex ( Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976 ) the 206-210 loop is shifted and now forms part of the so-called “new Pi” site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.

Identification of phosphorylation sites of equine β‐casein isoforms

Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro-column. This method turned out to be an efficient tool to separate the CPPs Arg(1)-Lys(34) and Glu(4)-Lys(34) from non-phosphorylated peptides. Purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) and each CPP was hydrolyzed by endoproteinase Glu-C. Finally, the digests were analyzed by RP-HPLC/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) and identified by nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) to locate the phosphorylated sites of the beta-casein isoforms 4P-7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser(9), Ser(23), Ser(24), and Ser(25). Addition of phosphate groups on Ser(18), Thr(12), and Ser(10) led to the formation of the isoforms 5P-7P, respectively. The results indicated that the in vivo phosphorylation of the equine beta-casein follows a sequential way and is not randomly performed.

Binding of divalent metal ions to 1-25 β-caseinophosphopeptide: an isothermal titration calorimetry study.

To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to β-CN(1-25)4P, a β-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of β-CN(1-25)4P binds two moles of Ca(2+), Mg(2+) or Zn(2+) under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K=4900-11,200M(-1)). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu(2+) did not bind to β-CN(1-25)4P at pH 8, despite its reported significant affinity towards β-casein and the 1-25 peptide at near-neutral pH.

Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.

One of the most striking features of several X-ray structures of CoA-independent ALDHs (aldehyde dehydrogenases) in complex with NAD(P) is the conformational flexibility of the NMN moiety. However, the fact that the rate of the acylation step is high in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of serine for invariant Thr244 and alanine for Lys178 result in a drastic decrease of the efficiency of hydride transfer which becomes rate-limiting. The crystal structure of the binary complex T244S GAPN-NADP shows that the absence of the beta-methyl group leads to a well-defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild-type. The approximately 0.6-unit increase in pK(app) of the catalytic Cys302 observed in the ternary complex for both mutated GAPNs is likely to be due to a slight difference in positioning of the nicotinamide ring relative to Cys302 with respect to the wild-type ternary complex. Taken together, the data support a critical role of the Thr244 beta-methyl group, held in position through a hydrogen-bond interaction between the Thr244 beta-hydroxy group and the epsilon-amino group of Lys178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

Crystallization and preliminary X-ray studies of the glutaredoxin from poplar in complex with glutathione

A monocysteinic mutant of poplar glutaredoxin (C30S) has been overproduced and purified. The protein has been crystallized in complex with glutathione using the hanging-drop vapour-diffusion technique in the presence of PEG 4000 as a precipitating agent. A native data set was collected at 1.55 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.7, b = 49.1, c = 104.8 A. Isomorphous crystals of a selenomethionine derivative were grown under the same conditions. Three data sets were collected at 1.73 A using the FIP synchrotron beamline at the ESRF. The positions of the Se atoms were determined and model rebuilding and refinement are in progress.

Crystallization and preliminary X-ray data of a bifunctional peroxiredoxin from poplar

Two variants (wild type and V152C mutant) of a bifunctional poplar peroxiredoxin have been overexpressed in Escherichia coli cells. The two recombinant enzymes were purified and crystallized using the hanging-drop vapour-diffusion technique. Data sets were collected to 1.62 and 2.48 A resolution using X-ray synchrotron-source radiation from two crystal forms of wild-type peroxiredoxin which belonged to the monoclinic space group P2(1) (with unit-cell parameters a = 59.26, b = 68.80, c = 75.71 A, beta = 93.45 degrees ) and to the orthorhombic space group P2(1)2(1)2 (with unit-cell parameters a = 64.70, b = 130.73, c = 35.59 A), respectively. Data were also collected to 2.17 A resolution using a home X-ray source from a V152C peroxiredoxin crystal which belongs to the triclinic space group (P1), with unit-cell parameters a = 36.65, b = 41.53, c = 58.06 A, alpha = 70.52, beta = 93.45, gamma = 64.31 degrees. Phases have been obtained using molecular replacement with the structure of human peroxiredoxin V (PDB code 1hd2) as a search model. Refinement of the structures is in progress.