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.     

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.     

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.     

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.     

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.

     

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.     

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.     

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.     

A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone-like activity of a chloroplast-localized small heat shock protein

The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced organisms. Recently, five PMSR‐like genes were identified in Arabidopsis thaliana, including one plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that was chloroplast‐localized and highly expressed in actively photosynthesizing tissue ( Sadanandom A et al., 2000 ). However, no endogenous substrate to the pPMSR was identified. Here we report that a set of highly conserved methionine residues in Hsp21, a chloroplast‐localized small heat shock protein, can become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR‐catalyzed reduction of Hsp21 methionine sulfoxides occurred on a minute time‐scale, was ultimately DTT‐dependent and led to recovery of Hsp21 conformation and chaperone‐like activity, both of which are lost upon methionine sulfoxidation ( Härndahl et al., 2001 ). These data indicate that one important function of pPMSR may be to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial for cellular resistance to oxidative stress.     

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.     

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.     

Structure-function relationship in an archaebacterial methionine sulphoxide reductase B

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.     

Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function.

Reactive oxygen and nitrogen intermediates can cause damage to many cellular components and have been implicated in a number of diseases. Cells have developed a variety of mechanisms to destroy these reactive molecules or repair the damage once it occurs. In proteins one of the amino acids most easily oxidized is methionine, which is converted to methionine sulfoxide. An enzyme, peptide methionine sulfoxide reductase (MsrA), catalyzes the reduction of methionine sulfoxide in proteins back to methionine. There is growing evidence that MsrA plays an important role in protecting cells against oxidative damage. This paper reviews the biochemical properties and biological role of MsrA.     

A Glycine soja methionine sulfoxide reductase B5a interacts with the Ca2+/CAM‐binding kinase GsCBRLK and activates ROS signaling under carbonate alkaline stress

Although research has extensively illustrated the molecular basis of plant responses to salt and high‐pH stresses, knowledge on carbonate alkaline stress is poor and the specific responsive mechanism remains elusive. We have previously characterized a Glycine soja Ca²⁺/CAM‐dependent kinase GsCBRLK that could increase salt tolerance. Here, we characterize a methionine sulfoxide reductase (MSR) B protein GsMSRB5a as a GsCBRLK interactor by using Y2H and BiFc assays. Further analyses showed that the N‐terminal variable domain of GsCBRLK contributed to the GsMSRB5a interaction. Y2H assays also revealed the interaction specificity of GsCBRLK with the wild soybean MSRB subfamily proteins, and determined that the BoxI/BoxII‐containing regions within GsMSRBs were responsible for their interaction. Furthermore, we also illustrated that the N‐terminal basic regions in GsMSRBs functioned as transit peptides, which targeted themselves into chloroplasts and thereby prevented their interaction with GsCBRLK. Nevertheless, deletion of these regions allowed them to localize on the plasma membrane (PM) and interact with GsCBRLK. In addition, we also showed that GsMSRB5a and GsCBRLK displayed overlapping tissue expression specificity and coincident expression patterns under carbonate alkaline stress. Phenotypic experiments demonstrated that GsMSRB5a and GsCBRLK overexpression in Arabidopsis enhanced carbonate alkaline stress tolerance. Further investigations elucidated that GsMSRB5a and GsCBRLK inhibited reactive oxygen species (ROS) accumulation by modifying the expression of ROS signaling, biosynthesis and scavenging genes. Summarily, our results demonstrated that GsCBRLK and GsMSRB5a interacted with each other, and activated ROS signaling under carbonate alkaline stress.     

Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism

Almost all forms of reactive oxygen species (ROS) oxidize methionine residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide. Because organisms contain methionine sulfoxide reductases (Msr's) that can catalyze the thioredoxin-dependent reduction of the sulfoxides back to methionine, it was proposed that the cyclic oxidation/reduction of methionine residues might serve as antioxidants to scavenge ROS, and also to facilitate the regulation of critical enzyme activities. We summarize here results of studies showing that organisms possess two different forms of Msr – namely, MsrA that catalyzes reduction of the S-isomer and MsrB that catalyzes the reduction of the R-isomer. Deletion of the msrA gene in mice leads to increased sensitivity to oxidative stress and to a decrease (40%) in the maximum lifespan. This suggests that elimination of both Msr's would have more serious consequences.     

Ectopic Expression of Maize Plastidic Methionine Sulfoxide Reductase ZmMSRB1 Enhances Salinity Stress Tolerance in Arabidopsis thaliana

Plant methionine sulfoxide reductases (MSRs) can repair oxidative damage done to intracellular proteins and, therefore, play an active role in the response to abiotic stress. However, the function of MSR homologs in maize has not been reported, to the best of our knowledge. In a previous study, we reported that ZmMSRB1 can be induced by salinity stress. In this study, we revealed that ZmMSRB1 is localized to chloroplasts and belongs to the MSRB sub-family. Characterization of an Arabidopsis thaliana msrb1 mutant and lines with ectopic expression of MSRB1 indicated that MSRB1 contributes to tolerance of salinity stress. Overexpression of ZmMSRB1 in Arabidopsis seedlings significantly decreased reactive oxygen species (ROS) accumulation by leading to the downregulation of ROS-generating genes and upregulation of ROS-scavenging genes, which resulted in a significant increase in ROS-scavenging protein activity. ZmMSRB1 overexpression was also found to enhance the expression of Salt Overly Sensitive genes, which maintain intracellular K⁺/Na⁺ balance. Furthermore, it resulted in the promotion of expression of key genes involved in glucose metabolism, increasing the soluble sugar content in the leaves. The ZmMSRB1 protein was observed to physically interact with glutathione S-transferase ZmGSTF8 in a yeast two-hybrid assay. GST catalyzes the conjugation of glutathione (GSH) to other compounds, counteracting oxidative damage to cells in vivo. When GSH synthesis was disrupted, the ZmMSRB1-induced response to salinity stress was partially impaired. Together, the findings of the present study indicate that maize MSRB1 promotes resistance to salinity stress by regulating Na⁺/K⁺ transport, soluble sugar content, and ROS levels in A. thaliana.

     

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.     

Redox‐regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H‐site flexibility

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X‐ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH‐binding site (G‐site) and a hydrophobic co‐substrate‐binding site (H‐site). At elevated H₂O₂ concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H‐site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox‐regulated enzyme.