Biochemical properties of poplar thioredoxin z

AbstractTrx-z is a chloroplastic thioredoxin, exhibiting a usual WCGPC active site, but whose biochemical properties are unknown. We demonstrate here that Trx-z supports the activity of several plastidial antioxidant enzymes, such as thiol-peroxidases and methionine sulfoxide reductases, using electrons provided by ferredoxin–thioredoxin reductase. Its disulfide reductase activity requires the presence of both active site cysteines forming a catalytic disulfide bridge with a midpoint redox potential of −251mV at pH7. These in vitro biochemical data suggest that, besides its decisive role in the regulation of plastidial transcription, Trx-z might also be involved in stress response

Probing the Surface of a Laccase for Clues towards the Design of Chemo-Enzymatic Catalysts

Systems featuring a multi-copper oxidase associated with transition-metal complexes can be used to perform oxidation reactions in mild conditions. Here, a strategy is presented for achieving a controlled orientation of a ruthenium–polypyridyl graft at the surface of a fungal laccase. Laccase variants are engineered with unique surface-accessible lysine residues. Distinct ruthenium–polypyridyl-modified laccases are obtained by the reductive alkylation of lysine residues precisely located relative to the T1 copper centre of the enzyme. In none of these hybrids does the presence of the graft compromise the catalytic efficiency of the enzyme on the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Furthermore, the efficiency of the hybrids in olefin oxidation coupled to the light-driven reduction of O2 is highly dependent on the location of the graft at the enzyme surface. Simulated RuII–CuII electron coupling values and distances fit well the observed reactivity and could be used to guide future hybrid designs.L.S. was the recipient of a MinistHre de l’Education Nationale fellowship. This study was supported by grants from the Agence Nationale de la Recherche (ANR-09-BLANC-0176 and ANR-15-CE07-0021-01) and from the Ministerio de EconomÍa, Industria y Competitividad (CTQ2016-79138-R). We thank Elise Courvoisier-Dezord from the Plateforme AVB (AMU): Analyse et Valorisation de la Biodiversit8 and Yolande Charmasson for help in the production of the recombinant enzymes, as well as Pascal Mansuelle and R8gine Lebrun from the Plateforme Prot8omique (CNRSAMU) for help in acquiring mass spectrometry data.Peer ReviewedPostprint (published version

Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological and pathophysiological conditions, but its use as a redox marker suffers from the lack of tools to detect and quantify MetO within cells. In this work, we created a pair of complementary stereospecific genetically-encoded mechanism-based ratiometric fluorescent sensors of MetO by inserting a circularly yellow fluorescent protein between yeast methionine sulfoxide reductases and thioredoxins. The two sensors, named MetSOx and MetROx for their ability to detect S and R-forms of MetO, respectively, were utilized for targeted analysis of protein oxidation, regulation and repair, as well as for monitoring MetO in bacterial and mammalian cells, analyzing compartment-specific changes in MetO, and examining responses to physiological stimuli

Methionine sulfoxide reductases: redox control of protein function and protection against oxidative stress.

International audienceMethionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases enzymes named methionine sulfoxide reductases (Msr) to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity. MsrA can act similarly on the free amino acid and oxidized proteins, whereas MsrB reduces efficiently only protein-bound MetO. Another type of thiol-oxidoreductase, the free-methionine-R-sulfoxide reductase (fRMsr), identified in prokaryotes and fungi, reduces the R diastereomer of the free amino acid MetO only. These MetO reducing enzymes are partners of thioredoxins and glutaredoxins, from which they receive reducing power. All these Msrs play important roles in the protection of organisms against oxidative stress through two main functions: i) the repair of oxidized proteins, and ii) an antioxidant function through reactive oxygen species scavenging by cyclic Met oxidation and reduction. Moreover, the reversible Met oxidation was shown to act as a post-translational modification responsible for the activation of enzymes and transcription factors or the regulation of protein-protein interactions. Some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. Among these, the periplasmic MsrP reduces efficiently protein-bound MetO without stereospecificity. After a journey in the Msr world, I will present you the latest results about the periplasmic systems of MetO reduction in the purple bacteria Rhodobacter sphaeroides

Recharging oxidative protein repair: Catalysis by methionine sulfoxide reductases towards their amino acid, protein, and model substrates

The sulfur-containing amino acid methionine (Met) in its free and amino acid residue forms can be readily oxidized to the R and S diastereomers of methionine sulfoxide (MetO). Methionine sulfoxide reductases A (MSRA) and B (MSRB) reduce MetO back to Met in a stereospecific manner, acting on the S and R forms, respectively. A third MSR type, fRMSR, reduces the R form of free MetO. MSRA and MSRB are spread across the three domains of life, whereas fRMSR is restricted to bacteria and unicellular eukaryotes. These enzymes protect against abiotic and biotic stresses and regulate lifespan. MSRs are thiol oxidoreductases containing catalytic redox-active cysteine or selenocysteine residues, which become oxidized by the substrate, requiring regeneration for the next catalytic cycle. These enzymes can be classified according to the number of redox-active cysteines (selenocysteines) and the strategies to regenerate their active forms by thioredoxin and glutaredoxin systems. For each MSR type, we review catalytic parameters for the reduction of free MetO, low molecular weight MetO-containing compounds, and oxidized proteins. Analysis of these data reinforces the concept that MSRAs reduce various types of MetO-containing substrates with similar efficiency, whereas MSRBs are specialized for the reduction of MetO in proteins

Physiological Roles of Plant Methionine Sulfoxide Reductases in Redox Homeostasis and Signaling

International audienceOxidation of methionine (Met) leads to the formation of two S- and R-diastereoisomersof Met sulfoxide (MetO) that are reduced back to Met by methionine sulfoxide reductases (MSRs),A and B, respectively. Here, we review the current knowledge about the physiological functionsof plant MSRs in relation with subcellular and tissue distribution, expression patterns, mutantphenotypes, and possible targets. The data gained from modified lines of plant models and cropspecies indicate that MSRs play protective roles upon abiotic and biotic environmental constraints.They also participate in the control of the ageing process, as shown in seeds subjected to adverseconditions. Significant advances were achieved towards understanding how MSRs could fulfil thesefunctions via the identification of partners among Met-rich or MetO-containing proteins, notably byusing redox proteomic approaches. In addition to a global protective role against oxidative damagein proteins, plant MSRs could specifically preserve the activity of stress responsive effectors suchas glutathione-S-transferases and chaperones. Moreover, several lines of evidence indicate thatMSRs fulfil key signaling roles via interplays with Ca$^{2+}$- and phosphorylation-dependent cascades,thus transmitting ROS-related information in transduction pathways

Les Méthionine sulfoxyde reductases de type B plastidiales d'Arabidopsis Thaliana (mécanismes de régénération par les thioredoxines et les glutaredoxines, cibles potentielles et rôles physiologiques)

Les espèces actives de l oxygène peuvent oxyder les protéines, au niveau d acides aminés sensibles comme la méthionine qui est convertie en deux diastéréoisomères, S et R, de méthionine sulfoxyde (MetSO). Cette modification est réversible par action des méthionine sulfoxyde réductases (MSR) de types A et B, spécifiques des diastéréoisomères S et R, respectivement. Les MSR utilisent généralement le pouvoir réducteur fourni par des oxydoréductases à thiol de type thiorédoxine (Trx) pour régénérer leur activité. La plante modèle Arabidopsis thaliana possède deux gènes codant des MSRB chloroplastiques, MSRB1 et MSRB2. Le travail réalisé au cours de cette thèse vise à caractériser les propriétés biochimiques et les rôles de ces deux isoformes. L existence d une spécificité marquée dans les systèmes régénérant l activité de ces MSR a été montrée. MSRB2 est réduite par les Trx des familles f, m et y selon un échange dithiol/disulfure alors que MSRB1 est régénérée par une Trx particulière, CDSP32, et par le système constitué du glutathion et des glutarédoxines (Grx). La régénération de l activité de MSRB1 s effectue par la réduction directe de l acide sulfénique produit sur la cystéine catalytique après réduction de la MetSO, par CDSP32 ou par le glutathion. La forme glutathionylée de MSRB1 est réduite par les Grx. La recherche des cibles des MSRB plastidiales a permis d identifier des protéines impliquées dans des processus fondamentaux, comme la photosynthèse, la traduction ou la protection contre le stress. L étude du phénotype de mutants d Arabidopsis thaliana, dont l expression des gènes MSRB1 et MSRB2 est supprimée, a montré que ces deux isoformes sont responsables de la majorité de l activité MSR dans les feuilles et qu elles sont impliquées dans le maintien de la croissance lors de contraintes environnementales par un rôle de protection de l appareil photosynthétique.Reactive oxygen species can alter protein properties through the oxidation of susceptible amino acids such as methionine. Methionine oxidation leads to the formation of two diastereoisomers, S and R, of methionine sulfoxide (MetSO). This modification is reversible thanks to the action of two types of methionine sulfoxide reductases (MSRs), A and B, specific to the diastereoisomers S and R, respectively. MSRs generally use thioredoxins (Trxs) as a source of electrons for regenerating their activity. The plant model Arabidopsis thaliana possesses two plastidial MSRBs, termed MSRB1 and MSRB2. The PhD work aims to characterize the biochemical properties and the roles of the two isoforms. Our data show a strong specificity in the systems regenerating the activity of these enzymes: MSRB2 is reduced only by Trxs f, m and y through a classical dithiol/disulfide exchange, whereas MSRB1 is regenerated by the unusual CDSP32 Trx and by the system composed of glutathione and glutaredoxins (Grxs). MSRB1 regeneration proceeds through the direct reduction by CDSP32 or by glutathione of the sulfenic acid formed on catalytic cysteine after MetSO reduction. The glutathionylated form of MSRB1 is subsequently reduced by Grxs. The search for potential targets of MSRBs has led to the identification of proteins involved in fundamental processes such as photosynthesis, translation or protection against stress. The characterization of Arabidopsis mutants defective for the expression of MSRB1 and MSRB2 genes has revealed that both enzymes account for most MSR activity in leaves and preserve plant growth during environmental constraints through a role in the protection of the photosynthetic apparatusAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Protein-Repairing Methionine Sulfoxide Reductases in Photosynthetic Organisms: Gene Organization, Reduction Mechanisms, and Physiological Roles

Methionine oxidation to methionine sulfoxide (MetSO) is reversed by two types of methionine sulfoxide reductases (MSRs), A and B, specific to the S- and R-diastereomers of MetSO, respectively. MSR genes are found in most organisms from bacteria to human. In the current review, we first compare the organization of the MSR gene families in photosynthetic organisms from cyanobacteria to higher plants. The analysis reveals that MSRs constitute complex families in higher plants, bryophytes, and algae compared to cyanobacteria and all non-photosynthetic organisms. We also perform a classification, based on gene number and structure, position of redox-active cysteines and predicted sub-cellular localization. The various catalytic mechanisms and potential physiological electron donors involved in the regeneration of MSR activity are then described. Data available from higher plants reveal that MSRs fulfill an essential physiological function during environmental constraints through a role in protein repair and in protection against oxidative damage. Taking into consideration the expression patterns of MSR genes in plants and the known roles of these genes in non-photosynthetic cells, other functions of MSRs are discussed during specific developmental stages and ageing in photosynthetic organisms

Distribution of methionine sulfoxide reductases in fungi and conservation of the freemethionine-R-sulfoxide reductase in multicellular eukaryotes

Methionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine- R -sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi