Redox State Controls Phase Separation of the Yeast Ataxin-2 Protein via Reversible Oxidation of Its Methionine-Rich Low-Complexity Domain.

Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.

Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis.

Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.

Methionine sulfoxide reductase 2 regulates Cvt autophagic pathway by altering the stability of Atg19 and Ape1 in Saccharomyces cerevisiae.

The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts in vivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.

Catalytic Mechanism of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A.

The oxidation of Met to methionine sulfoxide (MetSO) by oxidants such as hydrogen peroxide, hypochlorite, or peroxynitrite has profound effects on protein function. This modification can be reversed by methionine sulfoxide reductases (msr). In the context of pathogen infection, the reduction of oxidized proteins gains significance due to microbial oxidative damage generated by the immune system. For example, Mycobacterium tuberculosis (Mt) utilizes msrs (MtmsrA and MtmsrB) as part of the repair response to the host-induced oxidative stress. The absence of these enzymes makes Mycobacteria prone to increased susceptibility to cell death, pointing them out as potential therapeutic targets. This study provides a detailed characterization of the catalytic mechanism of MtmsrA using a comprehensive approach, including experimental techniques and theoretical methodologies. Confirming a ping-pong type enzymatic mechanism, we elucidate the catalytic parameters for sulfoxide and thioredoxin substrates (kcat/KM = 2656 ± 525 M-1 s-1 and 1.7 ± 0.8 × 106 M-1 s-1, respectively). Notably, the entropic nature of the activation process thermodynamics, representing ∼85% of the activation free energy at room temperature, is underscored. Furthermore, the current study questions the plausibility of a sulfurane intermediate, which may be a transition-state-like structure, suggesting the involvement of a conserved histidine residue as an acid-base catalyst in the MetSO reduction mechanism. This mechanistic insight not only advances our understanding of Mt antioxidant enzymes but also holds implications for future drug discovery and biotechnological applications.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides.

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Methionine oxidation in bacteria: A reversible post-translational modification.

Methionine is a sulfur-containing residue found in most proteins which are particularly susceptible to oxidation. Although methionine oxidation causes protein damage, it can in some cases activate protein function. Enzymatic systems reducing oxidized methionine have evolved in most bacterial species and methionine oxidation proves to be a reversible post-translational modification regulating protein activity. In this review, we inspect recent examples of methionine oxidation provoking protein loss and gain of function. We further speculate on the role of methionine oxidation as a multilayer endogenous antioxidant system and consider its potential consequences for bacterial virulence.

MSRB2 Ameliorates H2O2-induced Chondrocyte Oxidative Stress and Suppresses Apoptosis in Osteoarthritis.

BACKGROUND: Chondrocyte oxidative stress and apoptosis are critical factors contributing to the pathogenesis of osteoarthritis (OA). Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial protein that protects cells from oxidative stress and is involved in apoptosis. This study aimed to investigated the expression of MSRB2 in articular cartilage tissues and elucidated its effect on H2O2-stimulated chondrocytes. METHODS: Human chondrocytes were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12. MSRB2 overexpression in chondrocytes was achieved by transfecting with an MSRB2 overexpression plasmid. Western blot, quantitative RT-PCR, Immunofluorescence staining, and TUNEL assay were employed in this study. RESULTS: MSRB2 expression was found to be reduced in OA patients. Furthermore, overexpression of MSRB2 in H2O2-induced chondrocytes mitigated apoptosis and enhanced cell viability. Elevated MSRB2 expression diminished chondrocyte ROS contents, decreased cytochrome C (Cyc) in the cytoplasm, and regulated mitochondrial membrane potential to maintain mitochondrial homeostasis. Interestingly, knockdown of charged multivesicular body protein 5 (CHMP5) led to a decreased inMSRB2 expression in chondrocytes. Additionally, protein levels of CHMP5 and MSRB2 were reduced in H2O2-stimulated chondrocytes, and silencing CHMP5 reduced MSRB2 expression. Knockdown of CHMP5 increased cleaved caspase-3 expression in H2O2-induced chondrocytes and elevated TUNEL-positive chondrocytes. CONCLUSION: MSRB2 decreased in OA, and overexpression of MSRB2 alleviated oxidative stress and apoptosis of chondrocyte.

METHIONINE SULFOXIDE REDUCTASE A AND NEUROTRANSMISSION ENZYMES IN AUTISM SPECTRUM DISORDER AND DYSTOCIA RELATED AUTISTICS.

Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme that repairs the oxidation of methionine residues in proteins and free methionine in autism spectrum disorder (ASD). The present study aimed to assess the level of MsrA and neurotransmission enzymes in ASD individuals. Results confirmed that ASD associated with significant (P<0.05) reduction of MsrA and modulated mission enzymes. The role of MsrA as repair enzyme should be taken into account for study the activity of brain enzymes and proteins in ASD including ASMT that has a role in melatonin problems production in ASD due to higher AANAT level. The influence of MsrA also should be studied with MAT in mice to give more evidence.

You cannot oxidize what you cannot reach: Oxidative susceptibility of buried methionine residues.

Oxidation of protein methionines to methionine sulfoxides can result in protein structural alterations with a wide variety of biological implications. Factors that determine susceptibility to oxidation are not well understood. The recent JBC Editors Pick by Walker et al. applied proteomic methodologies to show that the oxidative susceptibility of buried methionine residues is strongly correlated with folding stability of the contextual peptide. Proteome-wide analysis of oxidation-susceptible methionines promises to answer open questions about the biological functions of reversible methionine oxidation.

Methionine oxidation under anaerobic conditions in Escherichia coli.

Repairing oxidative-targeted macromolecules is a central mechanism necessary for living organisms to adapt to oxidative stress. Reactive oxygen and chlorine species preferentially oxidize sulfur-containing amino acids in proteins. Among these amino acids, methionine can be converted into methionine sulfoxide. This post-translational oxidation can be reversed by methionine sulfoxide reductases, Msr enzymes. In Gram-negative bacteria, the antioxidant MsrPQ system is involved in the repair of periplasmic oxidized proteins. Surprisingly, in this study, we observed in Escherichia coli that msrPQ was highly expressed in the absence of oxygen. We have demonstrated that the anaerobic induction of msrPQ was due to chlorate (ClO3 - ) contamination of the Casamino Acids. Molecular investigation led us to determine that the reduction of chlorate to the toxic oxidizing agent chlorite (ClO2 - ) by the three nitrate reductases (NarA, NarZ, and Nap) led to methionine oxidation of periplasmic proteins. In response to this stress, the E. coli HprSR two-component system was activated, leading to the over-production of MsrPQ. This study, therefore, supports the idea that methionine oxidation in proteins is part of chlorate toxicity, and that MsrPQ can be considered as an anti-chlorate/chlorite defense system in bacteria. Finally, this study challenges the traditional view of the absence of Met-oxidation during anaerobiosis.

Actin reduction by MsrB2 is a key component of the cytokinetic abscission checkpoint and prevents tetraploidy.

Abscission is the terminal step of cytokinesis leading to the physical separation of the daughter cells. In response to the abnormal presence of lagging chromatin between dividing cells, an evolutionarily conserved abscission/NoCut checkpoint delays abscission and prevents formation of binucleated cells by stabilizing the cytokinetic intercellular bridge (ICB). How this bridge is stably maintained for hours while the checkpoint is activated is poorly understood and has been proposed to rely on F-actin in the bridge region. Here, we show that actin polymerization is indeed essential for stabilizing the ICB when lagging chromatin is present, but not in normal dividing cells. Mechanistically, we found that a cytosolic pool of human methionine sulfoxide reductase B2 (MsrB2) is strongly recruited at the midbody in response to the presence of lagging chromatin and functions within the ICB to promote actin polymerization there. Consistently, in MsrB2-depleted cells, F-actin levels are decreased in ICBs, and dividing cells with lagging chromatin become binucleated as a consequence of unstable bridges. We further demonstrate that MsrB2 selectively reduces oxidized actin monomers and thereby counteracts MICAL1, an enzyme known to depolymerize actin filaments by direct oxidation. Finally, MsrB2 colocalizes and genetically interacts with the checkpoint components Aurora B and ANCHR, and the abscission delay upon checkpoint activation by nuclear pore defects also depends on MsrB2. Altogether, this work reveals that actin reduction by MsrB2 is a key component of the abscission checkpoint that favors F-actin polymerization and limits tetraploidy, a starting point for tumorigenesis.

An Intramolecular Charge Transfer-Förster Resonance Energy Transfer Integrated Unimolecular Platform for Two-Photon Ratiometric Fluorescence Sensing of Methionine Sulfoxide Reductases in Live-Neurons and Mouse Brain Tissues.

Oxidative stress in organisms is a factor leading to a series of diseases including tumors and neurological disorders, while methionine sulfoxide reductases (Msrs) may provide an antioxidant and self-repair mechanism through redox cycles of methionine residues in proteins. Thus, it is important to understand the crucial role of Msrs in maintaining the redox homeostasis. However, it remains a great challenge for real-time and quantitative monitoring of Msrs in live systems due to the lack of appropriate sensing tools. Herein, a novel unimolecular platform integrating the intramolecular charge transfer (ICT) and Förster resonance energy transfer (FRET) dual mechanisms was successfully developed. By employing the highly specific Msrs-catalyzed reduction from the electron-withdrawing sulfoxide moiety in the probe to an electron-donating sulfide group, a synergistic ICT-FRET activation process was achieved, leading to a ratiometric fluorescence response toward Msrs with high selectivity, sensitivity, and accuracy. Moreover, benefiting from the favorable features, including mitochondria-targeting, near-infrared two-photon excitation, low cytotoxicity, good stability, and biocompatibility, the probe was successfully used for monitoring mitochondrial Msrs levels in live-neurons, and a positively correlated up-regulation of endogenous Msrs levels under O2•- stimulation was observed for the first time, confirming a Msrs-involved adaptive antioxidant mechanism in neurons. Furthermore, two-photon microscopic imaging of various regions in Alzheimer's disease (AD) mice brains revealed a down-regulated Msrs levels compared with that in normal brains, especially in the cornuammonis of the hippocampus region, which may in turn lead to an aggravation of AD pathogenesis due to the weakened antioxidant and self-repair capability of neurons.

A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase (msrA) Expression for Oxidative Stress Adaptation.

Reactive oxygen species (ROS) can cause destructive damage to biological macromolecules and protein dysfunction in bacteria. Methionine sulfoxide reductase (Msr) with redox-active Cys and/or seleno-cysteine (Sec) residues can restore physiological functions of the proteome, which is essential for oxidative stress tolerance of the extremophile Deinococcus radiodurans. However, the underlying mechanism regulating MsrA enzyme activity in D. radiodurans under oxidative stress has remained elusive. Here, we identified the function of MsrA in response to oxidative stress. msrA expression in D. radiodurans was significantly upregulated under oxidative stress. The msrA mutant showed a deficiency in antioxidative capacity and an increased level of dabsyl-Met-S-SO, indicating increased sensitivity to oxidative stress. Moreover, msrA mRNA was posttranscriptionally regulated by a small RNA, DsrO. Analysis of the molecular interaction between DsrO and msrA mRNA demonstrated that DsrO increased the half-life of msrA mRNA and then upregulated MsrA enzyme activity under oxidative stress compared to the wild type. msrA expression was also transcriptionally regulated by the DNA-repairing regulator DrRRA, providing a connection for further analysis of protein restoration during DNA repair. Overall, our results provide direct evidence that DsrO and DrRRA regulate msrA expression at two levels to stabilize msrA mRNA and increase MsrA protein levels, revealing the protective roles of DsrO signaling in D. radiodurans against oxidative stress. IMPORTANCE The repair of oxidized proteins is an indispensable function allowing the extremophile D. radiodurans to grow in adverse environments. Msr proteins and various oxidoreductases can reduce oxidized Cys and Met amino acid residues of damaged proteins to recover protein function. Consequently, it is important to investigate the molecular mechanism maintaining the high reducing activity of MsrA protein in D. radiodurans during stresses. Here, we showed the protective roles of an sRNA, DsrO, in D. radiodurans against oxidative stress. DsrO interacts with msrA mRNA to improve msrA mRNA stability, and this increases the amount of MsrA protein. In addition, we also showed that DrRRA transcriptionally regulated msrA gene expression. Due to the importance of DrRRA in regulating DNA repair, this study provides a clue for further analysis of MsrA activity during DNA repair. This study indicates that protecting proteins from oxidation is an effective strategy for extremophiles to adapt to stress conditions.

Methionine sulfoxide reductase B5 plays a key role in preserving seed vigor and longevity in rice (Oryza sativa).

Oxidation of methionine leads to the formation of methionine S-sulfoxide and methionine R-sulfoxide, which can be reverted by two types of methionine sulfoxide reductase (MSR): MSRA and MSRB. Though the role of MSR enzymes has been elucidated in various physiological processes, the regulation and role of MSR in seeds remains poorly understood. In this study, through molecular, biochemical, and genetic studies using seed-specific overexpression and RNAi lines of OsMSRB5 in Oryza sativa, we demonstrate the role of OsMSRB5 in maintaining seed vigor and longevity. We show that an age-induced reduction in the vigor and viability of seeds is correlated with reduced MSR activity and increased methionine sulfoxide (MetSO) formation. OsMSRB5 expression increases during seed maturation and is predominantly localized to the embryo. Further analyses on transgenic lines reveal the role of OsMSRB5 in modulating reactive oxygen species (ROS) homeostasis to preserve seed vigor and longevity. We show that ascorbate peroxidase and PROTEIN l-ISOASPARTYL METHYLTRANSFERASE undergo MetSO modification in seeds that affects their functional competence. OsMSRB5 physically interacts with these proteins and reverts this modification to facilitate their functions and preserve seed vigor and longevity. Our results thus illustrate the role of OsMSRB5 in preserving seed vigor and longevity by modulating ROS homeostasis in seeds.

Design, Synthesis, and Evaluation of Probes for Spatially Resolved Imaging of Enantioselective Sulfoxide Reductases.

A novel concept of nonhydrolytic enzyme sensing based on aggregation-induced emission is described. As a proof of principle, fluorogenic probes for methionine sulfoxide reductases have been developed. Changes in the polarity and electronic nature upon reduction of sulfoxide to sulfide are translated to the aggregation potential of the probe. The new probes enable sensitive and highly spatially resolved imaging of the enzymatic activity.

Characterization of a new selenoprotein methionine sulfoxide reductase from Haematococcus pluvialis and its antioxidant activity in response to high light intensity, hydrogen peroxide, glyphosate, and cadmium exposure.

Selenium incorporates into selenocysteine (Sec) which is a key component of selenoproteins implicated in antioxidant defense and redox homeostasis. Methionine sulfoxide reductases (Msr) play crucial roles in cellular defense against environmental stress. Whereas mammals have the MsrB selenoprotein form, unicellular organisms have MsrA. The Sec residue at the conserved catalytic sites of selenoprotein MsrA confers a metabolic advantage over the non-selenoprotein type MsrA. In the present study, the novel selenoprotein HpMsrA from Haematococcus pluvialis was cloned by the rapid amplification of cDNA ends and transformed into the model green alga Chlamydomonas reinhardtii. Alignment of homologs revealed the presence of the conserved catalytic domain GUFW and showed that the HpMsrA protein comprises Sec (U) at the N-terminus but no recycled Cys at the C-terminus. We studied the response of HpMsrA expression to selenite, high light intensity, hydrogen peroxide, cadmium nitrate, and glyphosate exposure via real-time quantitative PCR and enzyme activity analysis. The results demonstrated that HpMsrA protects cellular proteins against oxidative and environmental stressors. Compared with wild type C. reinhardtii, the transformant exhibited a superior antioxidant ability. The discoveries made herein shed light on the antioxidant physiology and environmental stress resistance mechanisms of the selenoproteins in microalgae. This information may aid in conducting environmental risk assessments of aquatic ecosystems involving microalgae known to respond rapidly and quantitatively to abiotic stress factors promoting excessive reactive oxygen species generation.

Recombinant Humanized IgG1 Antibody Protects against oxLDL-Induced Oxidative Stress and Apoptosis in Human Monocyte/Macrophage THP-1 Cells by Upregulation of MSRA via Sirt1-FOXO1 Axis.

Oxidized low-density lipoprotein (oxLDL)-induced oxidative stress and apoptosis are considered as critical contributors to cardiovascular diseases. Methionine sulfoxide reductase A (MSRA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. Here, we firstly provide evidence that recombinant humanized IgG1 antibody treatment upregulated the expression of MSRA in THP-1 cells to defend against oxLDL-induced oxidative stress and apoptosis. It was also observed that the upregulation of MSRA is regulated by the forkhead box O transcription factor (FOXO1), and the acetylation of FOXO1 increased when exposed to oxLDL but declined when treated with recombinant humanized IgG1 antibody. In addition, we identified that silent information regulator 1 (SIRT1) suppresses FOXO1 acetylation. Importantly, SIRT1 or FOXO1 deficiency impaired the anti-oxidative stress and anti-apoptotic effect of recombinant humanized IgG1 antibody. Together, our results suggest that recombinant humanized IgG1 antibody exerts its anti-oxidative stress and anti-apoptotic function by upregulation of MSRA via the Sirt1-FOXO1 axis.

Activity of the yeast cytoplasmic Hsp70 nucleotide-exchange factor Fes1 is regulated by reversible methionine oxidation.

Cells employ a vast network of regulatory pathways to manage intracellular levels of reactive oxygen species (ROS). An effectual means used by cells to control these regulatory systems are sulfur-based redox switches, which consist of protein cysteine or methionine residues that become transiently oxidized when intracellular ROS levels increase. Here, we describe a methionine-based oxidation event involving the yeast cytoplasmic Hsp70 co-chaperone Fes1. We show that Fes1 undergoes reversible methionine oxidation during excessively-oxidizing cellular conditions, and we map the site of this oxidation to a cluster of three methionine residues in the Fes1 core domain. Making use of recombinant proteins and a variety of in vitro assays, we establish that oxidation inhibits Fes1 activity and, correspondingly, alters Hsp70 activity. Moreover, we demonstrate in vitro and in cells that Fes1 oxidation is reversible and is regulated by the cytoplasmic methionine sulfoxide reductase Mxr1 (MsrA) and a previously unidentified cytoplasmic pool of the reductase Mxr2 (MsrB). We speculate that inactivation of Fes1 activity during excessively-oxidizing conditions may help maintain protein-folding homeostasis in a suboptimal cellular folding environment. The characterization of Fes1 oxidation during cellular stress provides a new perspective as to how the activities of the cytoplasmic Hsp70 chaperones may be attuned by fluctuations in cellular ROS levels and provides further insight into how cells use methionine-based redox switches to sense and respond to oxidative stress.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.