Arabidopsis thaliana plastidial methionine sulfoxide reductases B,MSRBs, account for most leaf peptide MSR activity and are essential for growth under environmental constraints through a role in the preservation of photosystem antennae

Methionine oxidation to methionine sulfoxide (MetSO) is reversed by two types of methionine sulfoxide reductases (MSRs), A and B, specific to MetSO S‐ and R‐diastereomers, respectively. Two MSRB isoforms, MSRB1 and MSRB2, are present in chloroplasts of Arabidopsis thaliana. To assess their physiological role, we characterized Arabidopsis mutants knockout for the expression of MSRB1, MSRB2 or both genes. Measurements of MSR activity in leaf extracts revealed that the two plastidial MSRB enzymes account for the major part of leaf peptide MSR capacity. Under standard conditions of light and temperature, plants lacking one or both plastidial MSRBs do not exhibit any phenotype, regarding growth and development. In contrast, we observed that the concomitant absence of both proteins results in a reduced growth for plants cultivated under high light or low temperature. In contrast, double mutant lines restored for MSRB2 expression display no phenotype. Under environmental constraints, the MetSO level in leaf proteins is higher in plants lacking both plastidial MSRBs than in Wt plants. The absence of plastidial MSRBs is associated with an increased chlorophyll a/b ratio, a reduced content of Lhca1 and Lhcb1 proteins and an impaired photosynthetic performance. Finally, we show that MSRBs are able to use as substrates, oxidized cpSRP43 and cpSRP54, the two main components involved in the targeting of Lhc proteins to the thylakoids. We propose that plastidial MSRBs fulfil an essential function in maintaining vegetative growth of plants during environmental constraints, through a role in the preservation of photosynthetic antennae.     

Heterogeneity and function of mammalian MSRs: enzymes for repair, protection and regulation

Methionine sulfoxide, the physiologically relevant oxidation product of methionine, is enzymatically reduced by peptide methionine sulfoxide reductases (MSRs). Two distinct classes of these enzymes, MSRA and MSRB, which selectively reduce the two methionine sulfoxide epimers, methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, are found in virtually all organisms. Mammals typically possess only one gene encoding MSRA, but at least three genes encoding MSRBs. These MSRs show distinct tissue- and subcellular expression patterns and may play specific functional roles. Susceptibility of some ion channels to reversible methionine oxidation suggests that MSRs have a regulatory role in cellular excitability. Some--if not all--MSRs protect cells and organisms against a variety of oxidative stress episodes, including those by hypoxia and reperfusion, and play a modulatory role in lifespan determination. More MSR-dependent physiological phenomena await to be discovered.     

Specificity of thioredoxins and glutaredoxins as electron donors to two distinct classes of Arabidopsis plastidial methionine sulfoxide reductases B

Methionine sulfoxide reductases (MSRs) A and B reduce methionine sulfoxide (MetSO) S- and R-diastereomers, respectively, back to Met using electrons generally supplied by thioredoxin. The physiological reductants for MSRBs remain unknown in plants, which display a remarkable variety of thioredoxins (Trxs) and glutaredoxins (Grxs). Using recombinant proteins, we show that Arabidopsis plastidial MSRB1 and MSRB2, which differ regarding the number of presumed redox-active cysteines, possess specific reductants. Most simple-module Trxs, especially Trx m1 and Trx y2, are preferential and efficient electron donors towards MSRB2, while the double-module CDSP32 Trx and Grxs can reduce only MSRB1. This study identifies novel types of reductants, related to Grxs and peculiar Trxs, for MSRB proteins displaying only one redox-active cysteine.     

Regeneration Mechanisms of Arabidopsis thaliana Methionine Sulfoxide Reductases B by Glutaredoxins and Thioredoxins

Methionine oxidation leads to the formation of S- and R-diastereomers of methionine sulfoxide (MetSO), which are reduced back to methionine by methionine sulfoxide reductases (MSRs) A and B, respectively. MSRBs are classified in two groups depending on the conservation of one or two redox-active Cys; 2-Cys MSRBs possess a catalytic Cys-reducing MetSO and a resolving Cys, allowing regeneration by thioredoxins. The second type, 1-Cys MSRBs, possess only the catalytic Cys. The biochemical mechanisms involved in activity regeneration of 1-Cys MSRBs remain largely elusive. In the present work we used recombinant plastidial Arabidopsis thaliana MSRB1 and MSRB2 as models for 1-Cys and 2-Cys MSRBs, respectively, to delineate the Trx- and glutaredoxin-dependent reduction mechanisms. Activity assays carried out using a series of cysteine mutants and various reductants combined with measurements of free thiols under distinct oxidation conditions and mass spectrometry experiments show that the 2-Cys MSRB2 is reduced by Trx through a dithiol-disulfide exchange involving both redox-active Cys of the two partners. Regarding 1-Cys MSRB1, oxidation of the enzyme after substrate reduction leads to the formation of a stable sulfenic acid on the catalytic Cys, which is subsequently glutathionylated. The deglutathionylation of MSRB1 is achieved by both mono- and dithiol glutaredoxins and involves only their N-terminal conserved catalytic Cys. This study proposes a detailed mechanism of the regeneration of 1-Cys MSRB activity by glutaredoxins, which likely constitute physiological reductants for this type of MSR.Proteins are prone to oxidative modifications due to the action of reactive oxygen species. Methionine (Met), one of the most susceptible amino acids to oxidation (1), is converted into methionine sulfoxide (MetSO),³ resulting in altered conformation and activity for many proteins (1). Methionine sulfoxide reductases (MSRs), which catalyze the reduction of MetSO back to Met, are present in most living organisms. MSRA, the first MSR isolated (2), is specific of the MetSO S-diastereomer and participates in protection against oxidative stress (3). A second MSR type, MSRB, which catalytically reduces the MetSO R-diastereomer, was identified later (4). MSRA and MSRB are monomeric enzymes that display no sequence or structural homologies but share a similar three-step catalytic mechanism, (i) reduction of MetSO by MSR and formation of a sulfenic acid intermediate on the “catalytic” cysteine (Cys), (ii) formation of a disulfide bond between catalytic and “resolving” Cys and release of H₂O, and (iii) reduction of the disulfide bond by a reductant (5, 6). Thioredoxins (Trxs) have been proposed to be the biological reductant for MSRs (2, 7). Trxs are small and ubiquitous disulfide reductases with a WC(G/P)PC active site. They function as electron donors and play essential roles in many processes through control of protein conformation and activity by supplying the reducing power needed to reduce disulfide bonds in target proteins.Most MSRBs, named 2-Cys MSRBs, possess two conserved Cys and are actually reduced by Trxs (7, 8). However, in a second class of MSRBs, termed 1-Cys MSRBs and representing ∼40% of known MSRBs, the resolving Cys residue corresponding to Cys-63 in Escherichia coli is replaced by Thr or Ser (8, 9). Although some of these MSRBs possess another potential resolving Cys (9), most 1-Cys MSRBs do not have any additional Cys, indicating that an alternative mechanism, which does not involve the formation of an intramolecular disulfide reduction, is needed for their regeneration (7). Contrasting data concerning the role of Trxs in providing electrons to these MSRBs have been reported. Several studies showed that cytosolic Trx is not an efficient reductant for human 1-Cys MSRBs (10–12), whereas mitochondrial Trxs were recently reported to efficiently regenerate mitochondrial 1-Cys MSRBs (13). It has been proposed that regeneration of mammalian and plant 1-Cys MSRBs could involve direct reduction of the cysteine sulfenic acid form generated during catalysis (10, 13–15).Arabidopsis thaliana possesses two plastidial MSRBs referred to as MSRB1 and MSRB2 and related to 1-Cys and 2-Cys MSRB types, respectively. MSRB2 possesses two CXXC motifs potentially implicated in the coordination of a zinc atom, a Cys in position 187 corresponding to the catalytic Cys-117 of E. coli MSRB, a potential resolving Cys in position 134, and an additional Cys in position 115. MSRB1 also contains the four Cys residues potentially coordinating zinc, the potential catalytic Cys-186, and a Cys in position 116, whereas the potential resolving Cys is replaced by a Thr in position 132. Previously, we showed that various types of canonical Trxs are efficient electron suppliers to MSRB2, whereas MSRB1 can only be reduced by the peculiar Trx CDSP32 (chloroplastic drought-induced stress protein of 32 kDa) and by Grxs (15–17). Grxs are oxidoreductases of the Trx superfamily possessing either a monothiol CXXS or a dithiol CXXC active site and are generally reduced by glutathione (18). Grxs are able to reduce protein disulfides, but also glutathione-mixed disulfides, a reaction termed deglutathionylation, for which Trxs are not efficient catalysts (19, 20). Classical dithiol Grxs can reduce disulfide bonds using both active site Cys residues, as shown for E. coli ribonucleotide reductase, but can also reduce glutathione-mixed disulfides through a monothiol mechanism that requires only the N-terminal active site Cys (21). CXXS-type Grxs catalyze deglutathionylation either through a monothiol mechanism, as recently shown for chloroplastic GrxS12 (CSYS active site) (22), or through a dithiol mechanism as suggested for Grxs with a CGFS active site (20, 23).We reported recently the involvement of Grxs in the regeneration of MSRB activity (15). Nevertheless, the precise biochemical mechanism underlying regeneration by Grxs remains unknown. In this study we performed a detailed analysis of the roles of redox-active Cys in reductants (Trxs and Grxs) and in acceptors (plastidial Arabidopsis MSRBs). We provide evidence that reduction of MSRB2 by Trxs is achieved through a classical dithiol-disulfide exchange. The data on MSRB1 reveal that 1-Cys MSRBs are regenerated by Grxs through a glutathionylation step of the catalytic Cys.     

Diversity of Plant Methionine Sulfoxide Reductases B and Evolution of a Form Specific for Free Methionine Sulfoxide

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H₂O₂-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

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.     

Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.     

Plant methionine sulfoxide reductase A and B multigenic families

Methionine oxidation to methionine sulfoxide (MetSo), which results in modification of activity and conformation for many proteins, is reversed by an enzyme present in most organisms and termed as methionine sulfoxide reductase (MSR). On the basis of substrate stereospecificity, two types of MSR, A and B, that do not share any sequence similarity, have been identified. In the present review, we first compare the multigenic MSR families in the three plant species for which the genome is fully sequenced: Arabidopsis thaliana, Oryza sativa, and Populus trichocarpa. The MSR gene content is larger in A. thaliana (five MSRAs and nine MSRBs) compared to P. trichocarpa (five MSRAs and four MSRBs) and O. sativa (four MSRAs and three MSRBs). A complete classification based on gene structure, sequence identity, position of conserved reactive cysteines and predicted subcellular localization is proposed. On the basis of in silico and experimental data originating mainly from Arabidopsis, we report that some MSR genes display organ-specific expression patterns and that those encoding plastidic MSRs are highly expressed in photosynthetic organs. We also show that the expression of numerous MSR genes is enhanced by environmental conditions known to generate oxidative stress. Thioredoxins (TRXs) constitute very likely physiological electron donors to plant MSR proteins for the catalysis of MetSO reduction, but the specificity between the numerous TRXs and methionine sulfoxide reductases (MSRs) present in plants remains to be investigated. The essential role of plant MSRs in protection against oxidative damage has been recently demonstrated on transgenic Arabidopsis plants modified in the content of cytosolic or plastidic MSRA.     

OsMSRA4.1 and OsMSRB1.1, two rice plastidial methionine sulfoxide reductases,are involved in abiotic stress responses

In proteins, methionine residues are especially sensitive to oxidation, leading to the formation of S- and R-methionine sulfoxide diastereoisomers, and these two methionine sulfoxides can be specifically reversed by two types of methionine sulfoxide reductases (MSRs), MSRA and MSRB. Previously, we have identified a gene encoding a putative MSR from NaCl-treated roots of Brazilian upland rice (Oryza sativa L. cv. IAPAR 9) via subtractive suppression hybridization (Wu et al. in Plant Sci 168:847–853, 2005). Blast database analysis indicated that at least four MSRA and three MSRB orthologs exist in rice, and two of them, OsMSRA4.1 and OsMSRB1.1, were selected for further functional analysis. Expression analysis showed that both OsMSRA4.1 and OsMSRB1.1 are constitutively expressed in all organs and can be induced by various stress conditions. Subcellular localization and in vitro activity assay revealed that both OsMSR proteins are targeted to the chloroplast and have MSR activity. Overexpression of either OsMSRA4.1 or OsMSRB1.1 in yeast enhanced cellular resistance to oxidative stress. In addition, OsMSRA4.1-overexpressing transgenic rice plants also showed enhanced viability under salt treatment. Our results provide genetic evidence of the involvement of OsMSRs in the plant stress responses. X. Guo and Y. Wu contributed equally to this work.     

Cassava <Emphasis Type="Italic">C-repeat binding factor 1</Emphasis> gene responds to low temperature and enhances cold tolerance when overexpressed in Arabidopsis and cassava

Key message

Reactive oxygen species (ROS) oxidize methionine to methionine sulfoxide (MetSO) and thereby inactivate proteins. Methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Our results show that Arabidopsis thaliana MSR enzyme coding gene MSRB8 is required for effector-triggered immunity and containment of stress-induced cell death in Arabidopsis.

Abstract

Plants activate pattern-triggered immunity (PTI), a basal defense, upon recognition of evolutionary conserved molecular patterns present in the pathogens. Pathogens release effector molecules to suppress PTI. Recognition of certain effector molecules activates a strong defense, known as effector-triggered immunity (ETI). ETI induces high-level accumulation of reactive oxygen species (ROS) and hypersensitive response (HR), a rapid programmed death of infected cells. ROS oxidize methionine to methionine sulfoxide (MetSO), rendering several proteins nonfunctional. The methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Though a few plant MSR genes are known to provide tolerance against oxidative stress, their role in plant–pathogen interaction is not known. We report here that activation of cell death by avirulent pathogen or UV treatment induces expression of MSRB7 and MSRB8 genes. The T-DNA insertion mutant of MSRB8 exaggerates HR-associated and UV-induced cell death and accumulates a higher level of ROS than wild-type plants. The negative regulatory role of MSRB8 in HR is further supported by amiRNA and overexpression lines. Mutants and overexpression lines of MSRB8 are susceptible and resistant respectively, compared to the wild-type plants, against avirulent strains of Pseudomonas syringae pv. tomato DC3000 (Pst) carrying AvrRpt2, AvrB, or AvrPphB genes. However, the MSRB8 gene does not influence resistance against virulent Pst or P. syringae pv. maculicola (Psm) pathogens. Our results altogether suggest that MSRB8 function is required for ETI and containment of stress-induced cell death in Arabidopsis.

     

Different B-Type Methionine Sulfoxide Reductases in Chlamydomonas May Protect the Alga against High-Light, Sulfur-Depletion, or Oxidative Stress

The genome of unicellular green alga Chlamydomonas reinhardtii contains four genes encoding B-type methionine sulfoxide reductases, MSRBI.1, MSRB1.2, MSRB2.1, and MSRB2.2, with functions largely unknown. To understand the cell defense system mediated by the methionine suifoxide reductases in Chlamydomonas, we analyzed expression and physiological roles of the MSRBs under different abiotic stress conditions using immunoblotting and quantitative polymerase chain reaction （PCR） analyses. We showed that the MSRB2.2 protein was accumulated in cells treated with high light （1,300 μE-/m2 per s）, whereas MSRBI.1 was accumulated in the cells under 1 mmol/L H2O2 treatment or sulfur depletion. We observed that the cells with the MSRB2.2 knockdown and overexpression displayed increased and decreased sensitivity to high light, respectively, based on in situ chlorophyll a fluorescence measures. We also observed that the cells with the MSRBI.1 knockdown and overexpression displayed decreased and increased tolerance to sulfur-depletion and oxidative stresses, respectively, based on growth and H2- producing performance. The physiological implications revealed from the experimental data highlight the importance of MSRB2.2 and MSRBI.1 in protecting Chlamydomonas cells against adverse conditions such as high-light, sulfur-depletion, and oxidative stresses.     

Crystal structure of the Escherichia coli peptide methionine sulphoxide reductase at 1.9 A resolution

BACKGROUND: Peptide methionine sulphoxide reductases catalyze the reduction of oxidized methionine residues in proteins. They are implicated in the defense of organisms against oxidative stress and in the regulation of processes involving peptide methionine oxidation/reduction. These enzymes are found in numerous organisms, from bacteria to mammals and plants. Their primary structure shows no significant similarity to any other known protein. RESULTS: The X-ray structure of the peptide methionine sulphoxide reductase from Escherichia coli was determined at 3 A resolution by the multiple wavelength anomalous dispersion method for the selenomethionine-substituted enzyme, and it was refined to 1.9 A resolution for the native enzyme. The 23 kDa protein is folded into an alpha/beta roll and contains a large proportion of coils. Among the three cysteine residues involved in the catalytic mechanism, Cys-51 is positioned at the N terminus of an alpha helix, in a solvent-exposed area composed of highly conserved amino acids. The two others, Cys-198 and Cys-206, are located in the C-terminal coil. CONCLUSIONS: Sequence alignments show that the overall fold of the peptide methionine sulphoxide reductase from E. coli is likely to be conserved in many species. The characteristics observed in the Cys-51 environment are in agreement with the expected accessibility of the active site of an enzyme that reduces methionine sulphoxides in various proteins. Cys-51 could be activated by the influence of an alpha helix dipole. The involvement of the two other cysteine residues in the catalytic mechanism requires a movement of the C-terminal coil. Several conserved amino acids and water molecules are discussed as potential participants in the reaction.     

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.     

The Arabidopsis plastidic methionine sulfoxide reductase B proteins. Sequence and activity characteristics, comparison of the expression with plastidic methionine sulfoxide reductase A, and induction by photooxidative stress

Two types of methionine (Met) sulfoxide reductases (Msr) catalyze the reduction of Met sulfoxide (MetSO) back to Met. MsrA, well characterized in plants, exhibits an activity restricted to the Met-S-SO-enantiomer. Recently, a new type of Msr enzyme, called MsrB, has been identified in various organisms and shown to catalytically reduce the R-enantiomer of MetSO. In plants, very little information is available about MsrB and we focused our attention on Arabidopsis (Arabidopsis thaliana) MsrB proteins. Searching Arabidopsis genome databases, we have identified nine open reading frames encoding proteins closely related to MsrB proteins from bacteria and animal cells. We then analyzed the activity and abundance of the two chloroplastic MsrB proteins, MsrB1 and MsrB2. Both enzymes exhibit an absolute R-stereospecificity for MetSO and a higher catalytic efficiency when using protein-bound MetSO as a substrate than when using free MetSO. Interestingly, we observed that MsrB2 is reduced by thioredoxin, whereas MsrB1 is not. This feature of MsrB1 could result from the lack of the catalytical cysteine (Cys) corresponding to Cys-63 in Escherichia coli MsrB that is involved in the regeneration of Cys-117 through the formation of an intramolecular disulfide bridge followed by thioredoxin reduction. We investigated the abundance of plastidial MsrA and B in response to abiotic (water stress, photooxidative treatment) and biotic (rust fungus) stresses and we observed that MsrA and B protein levels increase in response to the photooxidative treatment. The possible role of plastidic MsrB in the tolerance to oxidative damage is discussed.     

Increased catalytic efficiency following gene fusion of bifunctional methionine sulfoxide reductase enzymes from Shewanella oneidensis

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.     

Solution structure and backbone dynamics of the reduced form and an oxidized form of E. coli methionine sulfoxide reductase A (MsrA): structural insight of the MsrA catalytic cycle

Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (MetSO)-containing proteins, back to methionine (Met). MsrAs are stereospecific for the S epimer whereas MsrBs reduce the R epimer of MetSO. Although structurally unrelated, the Msrs characterized so far display a similar catalytic mechanism with formation of a sulfenic intermediate on the catalytic cysteine and a concomitant release of Met, followed by formation of at least one intramolecular disulfide bond (between the catalytic and a recycling cysteine), which is then reduced by thioredoxin. In the case of the MsrA from Escherichia coli, two disulfide bonds are formed, i.e. first between the catalytic Cys51 and the recycling Cys198 and then between Cys198 and the second recycling Cys206. Three crystal structures including E. coli and Mycobacterium tuberculosis MsrAs, which, for the latter, possesses only the unique recycling Cys198, have been solved so far. In these structures, the distances between the cysteine residues involved in the catalytic mechanism are too large to allow formation of the intramolecular disulfide bonds. Here structural and dynamical NMR studies of the reduced wild-type and the oxidized (Cys51-Cys198) forms of C86S/C206S MsrA from E. coli have been carried out. The mapping of MetSO substrate-bound C51A MsrA has also been performed. The data support (1) a conformational switch occurring subsequently to sulfenic acid formation and/or Met release that would be a prerequisite to form the Cys51-Cys198 bond and, (2) a high mobility of the C-terminal part of the Cys51-Cys198 oxidized form that would favor formation of the second Cys198-Cys206 disulfide bond.     

Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase

Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met₁₀₁ and Met₁₃₃, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met₁₀₁ and Met₁₃₃ residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.