Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii reveals the unique properties of a chloroplastic CGFS-type glutaredoxin.

Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use GSH as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of approximately 30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX, which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase, and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (-323 +/- 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRXs proved to be very efficient catalysts of A(4)-glyceraldehyde-3-phosphate dehydrogenase deglutathionylation, whereas cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A(4)-glyceraldehyde-3-phosphate dehydrogenase is the first physiological substrate identified for a CGFS-type GRX

Ribulose-1,5-bisphosphate regeneration in the Calvin-Benson-Bassham cycle: Focus on the last three enzymatic steps that allow the formation of Rubisco substrate

The Calvin-Benson-Bassham (CBB) cycle comprises the metabolic phase of photosynthesis and is responsible for carbon fixation and the production of sugar phosphates. The first step of the cycle involves the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which catalyzes the incorporation of inorganic carbon into 3-phosphoglyceric acid (3PGA). The following steps include ten enzymes that catalyze the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco. While it is well established that Rubisco activity acts as a limiting step of the cycle, recent modeling studies and experimental evidence have shown that the efficiency of the pathway is also impacted by the regeneration of the Rubisco substrate itself. In this work, we review the current understanding of the structural and catalytic features of the photosynthetic enzymes that catalyze the last three steps of the regeneration phase, namely ribose-5-phosphate isomerase (RPI), ribulose-5-phosphate epimerase (RPE), and phosphoribulokinase (PRK). In addition, the redox- and metabolic-based regulatory mechanisms targeting the three enzymes are also discussed. Overall, this review highlights the importance of understudied steps in the CBB cycle and provides direction for future research aimed at improving plant productivity

Structural basis for the magnesium-dependent activation of transketolase from Chlamydomonas reinhardtii

Background In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg2 +. Methods TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography. Results CrTK is homodimeric and its catalysis depends on the reconstitution of the holo-enzyme in the presence of both TPP and Mg2 +. Activity measurements and CD analysis revealed that the formation of fully active holo-CrTK is Mg2 +-dependent and proceeds with a slow kinetics. The 3Dâstructure of CrTK without cofactors (CrTKapo) shows that two portions of the active site are flexible and disordered while they adopt an ordered conformation in the holo-form. Oxidative treatments revealed that Mg2 +participates in the redox control of CrTK by changing its propensity to be inactivated by oxidation. Indeed, the activity of holo-form is unaffected by oxidation whereas CrTK in the apo-form or reconstituted with the sole TPP show a strong sensitivity to oxidative inactivation. Conclusion These evidences indicate that Mg2 +is fundamental to allow gradual conformational arrangements suited for optimal catalysis. Moreover, Mg2 +is involved in the control of redox sensitivity of CrTK. General significance The importance of Mg2 +in the functionality and redox sensitivity of CrTK is correlated to light-dependent fluctuations of Mg2 +in chloroplasts

Contenuto corso Produttività Primaria e Sviluppo Sostenibile 2016/2017

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Thioredoxin and Glutaredoxin Systems

This Special Issue features recent data concerning thioredoxins and glutaredoxins from various biological systems, including bacteria, mammals, and plants. Four of the sixteen articles are review papers that deal with the regulation of development of the effect of hydrogen peroxide and the interactions between oxidants and reductants, the description of methionine sulfoxide reductases, detoxification enzymes that require thioredoxin or glutaredoxin, and the response of plants to cold stress, respectively. This is followed by eleven research articles that focus on a reductant of thioredoxin in bacteria, a thioredoxin reductase, and a variety of plant and bacterial thioredoxins, including the m, f, o, and h isoforms and their targets. Various parameters are studied, including genetic, structural, and physiological properties of these systems. The redox regulation of monodehydroascorbate reductase, aminolevulinic acid dehydratase, and cytosolic isocitrate dehydrogenase could have very important consequences in plant metabolism. Also, the properties of the mitochondrial o-type thioredoxins and their unexpected capacity to bind iron–sulfur center (ISC) structures open new developments concerning the redox mitochondrial function and possibly ISC assembly in mitochondria. The final paper discusses interesting biotechnological applications of thioredoxin for breadmaking

Thioredoxin and Glutaredoxin Systems Antioxidants Special Issue

The special issue on Thioredoxin and Glutaredoxin systems (https://www.mdpi.com/journal/antioxidants/special_issues/Thioredoxin_and_Glutaredoxin_Systems) was initiated in response to solicitations from Antioxidants after discussing with colleagues at two successive redox meetings sponsored by European Molecular Biology Organization (EMBO) and held in July 2017 in Moscow/St. Petersburg (http://redox.vub.ac.be/events/embo-redox-biology-conference.html) and in September of the same year in San Feliu de Guixols (Spain) (http://meetings.embo.org/event/17-thiol-ox).We could then submit the idea to long time collaborators and redox friends but also to other colleagues with whom we had the chance to get in touch with at these meetings. In general, although Antioxidants is a rather recent creation and its credentials were at the time not so well known, the idea of participating in a special issue was very well received and many of the contacted colleagues have answered positively.Of course, as our background is in plant sciences, this special issue mostly contains papers dealing with oxygenic phototrophs but other experimental model organisms are also addressed (bacteria, mammals, zebrafish, etc.). Overall the special issue contains 16 papers, 12 of those reporting experimental research data, and 4 others being more review-like although some of them also contain original bioinformatics data