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.     

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.     

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.     

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.     

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.     

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.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Abstract

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-Rsulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-R-sulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

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.     

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.

     

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.

     

Functioning methionine sulfoxide reductases A and B are present in human epidermal melanocytes in the cytosol and in the nucleus

Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for the first time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

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.     

Transport and utilization of methionine sulfoxide in the rabbit

Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na⁺-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na⁺ in the external medium. Increasing the Na⁺ gradient reduces the apparent K_t for methionine sulfoxide without causing any change in V_max. With an outward K⁺ gradient (vesicle > medium), valinomycin stimulates the Na⁺-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol > NADPH > glutathione > NADH.     

Molecular Evolution of Peptide Methionine Sulfoxide Reductases (MsrA and MsrB): On the Early Development of a Mechanism That Protects Against Oxidative Damage

Methionine sulfoxide reductases, enzymes that reverse the oxidation of methionine residues, have been described in a wide range of species. The reduction of the diastereoisomers of oxidized methionine is catalyzed by two different monomeric methionine sulfoxide reductases (MsrA and MsrB) and is best understood as an evolutionary response to high levels of oxygen either in the Earth’s atmosphere or possibly in more localized environments. Phylogenetic analyses of these proteins suggest that their distribution is the outcome of a complex history including many paralogy and lateral gene transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Decrease in Peptide Methionine Sulfoxide Reductase in Alzheimer''s Disease Brain

Previous studies have shown that the pathophysiology of Alzheimer's disease (AD) is linked to oxidative stress. Oxidative damage to different biomolecular components of the brain is a characteristic feature of AD. Recent evidence suggests that methionine may act as an antioxidant defense molecule in proteins by its ability to scavenge oxidants and, in the process, undergo oxidation to form methionine sulfoxide. The enzyme peptide, methionine sulfoxide reductase (MsrA), reverses methionine sulfoxide back to methionine, which once again is able to scavenge oxidants. The purpose of this study was to measure the activity of MsrA in the brain of AD patients compared with control subjects. Our results showed that there was a decline in MsrA activity in all brain regions studied in AD and this decline reached statistical significance in the superior and middle temporal gyri (p < 0.001), inferior parietal lobule (p < 0.05), and the hippocampus (p < 0.05) in AD. An elevation of protein carbonyl content was found in all brain regions except the cerebellum in AD and reached statistical significance in the superior and middle temporal gyri and hippocampus. Messenger RNA analysis suggests that the loss in enzyme activity may be the result of a posttranslational modification of MsrA or a defect of translation resulting in inferior processing of the MsrA mRNA. Our results suggest that a decline in MsrA activity could reduce the antioxidant defenses and increase the oxidation of critical proteins in neurons in the brain in AD.     

New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli

It is known that reactive oxygen species can oxidize methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage. MsrA and MsrB are members of the methionine sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of methionine sulfoxide in proteins. However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of methionine sulfoxide in proteins. Employing a msrA/B double mutant, a new peptide methionine sulfoxide reductase activity has been found associated with membrane vesicles from E. coli. Both the R and S forms of N-acetylmethionine sulfoxide, D-ala-met(o)-enkephalin and methionine sulfoxide, are reduced by this membrane associated activity. The reaction requires NADPH and may explain, in part, how the R form of methionine sulfoxide in proteins is reduced in E. coli. In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.