Engineering of methionine sulfoxide reductase A with simultaneously improved stability and activity for kinetic resolution of chiral sulfoxides.

Methionine sulfoxide reductase A (MsrA) has emerged as promising biocatalysts in the enantioselective kinetic resolution of racemic (rac) sulfoxides. In this study, we engineered robust MsrA variants through directed evolution, demonstrating substantial improvements of thermostability. Mechanism analysis reveals that the enhanced thermostability results from the strengthening of intracellular interactions and increase in molecular compactness. Moreover, these variants demonstrated concurrent improvements in catalytic activities, and notably, these enhancements in stability and activity collectively contributed to a significant improvement in enzyme substrate tolerance. We achieved kinetic resolution on a series of rac-sulfoxides with high enantioselectivity under initial substrate concentrations reaching up to 93.0 g/L, representing a great improvement in the aspect of the substrate concentration for biocatalytic preparation of chiral sulfoxide. Hence, the simultaneously improved thermostability, activity and substrate tolerance of MsrA represent an excellent biocatalyst for the green synthesis of optically pure sulfoxides.

Biochemical characterization of GAF domain of free-R-methionine sulfoxide reductase from Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.

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.

Tomato methionine sulfoxide reductase B2 functions in drought tolerance by promoting ROS scavenging and chlorophyll accumulation through interaction with Catalase 2 and RBCS3B.

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of common metabolism and as the result of defense and development. ROS readily oxidizes methionine (Met) residues of proteins to form Met-R-sulfoxide or Met-S-sulfoxide (MetSO), resulting in protein inactivation or malfunction. Although it is known that MetSO can be reverted to Met by methionine sulfoxide reductase (Msr), the mechanism how Msr interacts with its target proteins is poorly understood. In this study, two target proteins of tomato MsrB2 (SlMsrB2), catalase 2 (CAT2) and the Rubisco small subunit RBCS3B, were identified. Silencing of SlMsrB2 by RNA interference (RNAi) in tomato led to decreased drought tolerance, accompanied by increased ROS accumulation and chlorophyll degradation. By contrast, overexpression of SlMsrB2 in tomato significantly reduced ROS accumulation and enhanced drought tolerance. Protein interaction analysis showed that SlMsrB2 interacts with CAT2 and RBCS3B in vitro and in planta. Silencing of CAT2 by RNAi and RBCS3B by virus-induced gene silencing (VIGS) resulted in development of pale green leaves and enhanced ROS accumulation in tomato plants. These results demonstrate that SlMsrB2 functions in drought tolerance and promotes chlorophyll accumulation by modulating ROS accumulation.

Functional characterization of methionine sulfoxide reductases from Leptospira interrogans.

BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.

Molecular Expression of Bioactive Recombinant Methionine Sulfoxide Reductase A (MsrA).

BACKGROUND: The increase in reactive oxygen species (ROS) production during cryopreservation of semen, leads to oxidation of biomolecules affecting the functionality of spermatozoa. Methionine residues in proteins are highly prone to oxidation and get converted into methionine sulfoxide (MetO). Methionine sulfoxide reductase A (MsrA) can improve the functionality of spermatozoa by reducing the MetO to methionine restoring the lost functionality of the affected proteins. OBJECTIVE: The expression of catalytically active recombinant MsrA (rMsrA). METHODS: The msrA gene was PCR amplified, cloned and sequenced. Further, the recombinant clone was used for protein expression and purification. The protein was getting precipitated during dialysis in Tris-buffer. Hence, the purified rMsrA was dialyzed at 4°C against the Tris-buffer pH 7.5 containing MgCl2, KCl, NaCl, urea and triton X-100. During dialysis, changes of buffer were done at every 12 h interval with stepwise reduction in the concentrations of NaCl, urea and triton X-100. The final dialysis was done with buffer containing 10 mM MgCl2, 30 mM KCl, and 150 mM NaCl, 25 mM Tris-HCl pH 7.5. The activity of the rMsrA was checked spectrophotometrically. RESULTS: The protein BLAST of buffalo MsrA with bovine sequence showed 14 amino acid mismatches. The rMsrA has been purified under denaturing conditions as it was forming inclusion bodies consistently during protein expression. After renaturation, the purified 33 kDa rMsrA was catalytically active by biochemical assay. CONCLUSION: The rMsrA expressed in prokaryotic system is catalytically active and can be used for supplementation to semen extender to repair the oxidatively damaged seminal plasma proteins that occur during cryopreservation.

Implication of Staphylococcus aureus MsrB dimerization upon oxidation.

Oxidative modification of protein structure has been shown to play a significant role in bacterial virulence and metabolism. The sulfur-containing residues are susceptible to oxidation and the enzymatic reversal of oxidized cysteine or methionine is detected in many organisms. Methionine sulfoxide reductases (Msr) are responsible for reducing oxidized methionine. The two different Msrs, MsrA and MsrB, reduce methionine R-sulfoxide and methionine S-sulfoxide, respectively through self-oxidation. This study elucidated the structure of MsrB from Staphylococcus aureus Mu50 and its changes upon oxidation. The active site shows two reduced cysteines in a close contact, implying disulfide bond would form without major structural rearrangement. When the protein is exposed to an oxidative condition, a dimeric state is observed. The dimerization of SAMsrB creates a valley structure for accepting peptidyl substrates. To the best of our knowledge, oxidation induced dimerization of SAMsrB would help to understand mechanism behind redox control that has not been well characterized.

Structure and Electron-Transfer Pathway of the Human Methionine Sulfoxide Reductase MsrB3.

Aims: The post-translational oxidation of methionine to methionine sulfoxide (MetSO) is a reversible process, enabling the repair of oxidative damage to proteins and the use of sulfoxidation as a regulatory switch. MetSO reductases catalyze the stereospecific reduction of MetSO. One of the mammalian MetSO reductases, MsrB3, has a signal sequence for entry into the endoplasmic reticulum (ER). In the ER, MsrB3 is expected to encounter a distinct redox environment compared with its paralogs in the cytosol, nucleus, and mitochondria. We sought to determine the location and arrangement of MsrB3 redox-active cysteines, which may couple MsrB3 activity to other redox events in the ER. Results: We determined the human MsrB3 structure by using X-ray crystallography. The structure revealed that a disulfide bond near the protein amino terminus is distant in space from the active site. Nevertheless, biochemical assays showed that these amino-terminal cysteines are oxidized by the MsrB3 active site after its reaction with MetSO. Innovation: This study reveals a mechanism to shuttle oxidizing equivalents from the primary MsrB3 active site toward the enzyme surface, where they would be available for further dithiol-disulfide exchange reactions. Conclusion: Conformational changes must occur during the MsrB3 catalytic cycle to transfer oxidizing equivalents from the active site to the amino-terminal redox-active disulfide. The accessibility of this exposed disulfide may help couple MsrB3 activity to other dithiol-disulfide redox events in the secretory pathway.

Development of a simple high-throughput assay for directed evolution of enantioselective sulfoxide reductases.

We report on the development of high-throughput fluorogenic assay that can streamline directed evolution of enantioselective sulfoxide reductases. As a model, methionine sulfoxide reductase A (MsrA) has been evolved to expand its limited substrate scope. The resulting mutant MsrA can resolve a range of new challenging racemic sulfoxides with high efficiency including the pharmaceutically relevant albendazole sulfoxide. The simplicity and the level of throughput make this method also suitable for the screening of metagenomic libraries in future for the discovery of new enzymes with similar reactivities.

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.

Enzymatic kinetic resolution of chiral sulfoxides - an enantiocomplementary approach.

A new enzymatic assay for the preparation of chiral sulfoxides that is enantiocomplementary to the known (S)-enantiomer-reducing activity of methionine sulfoxide reductase A (MsrA) is described. To this end, we have utilized the enzyme DMSO reductase (DmsABC), recently discovered by us being highly upregulated in stationary phase E. coli bacteria.

Resolving oxidative damage to methionine by an unexpected membrane-associated stereoselective reductase discovered using chiral fluorescent probes.

Nonenzymatic oxidative processes in living organisms are among the inevitable consequences of respiration and environmental conditions. These oxidative processes can lead to the formation of two stereoisomers (R and S) of methionine sulfoxide, and the redox balance between methionine and methionine sulfoxide in proteins has profound implications on their function. Methionine oxidation can be reverted enzymatically by methionine sulfoxide reductases (Msrs). The two enzyme classes known to fulfill this role are MsrA, reducing the (S)-isomer, and MsrB, reducing the (R)-isomer of methionine sulfoxide. They are strictly stereoselective and conserved throughout the tree of life. Under stress conditions such as stationary phase and nutrient starvation, Escherichia coli upregulates the expression of MsrA but a similar effect has not been described for MsrB, raising the conundrum of which pathway enables reduction of the (R)-isomer of methionine sulfoxide in these conditions. Using the recently developed chiral fluorescent probes Sulfox-1, we show that in stationary phase-stressed E. coli, MsrA does have a stereocomplementary activity reducing the (R)-isomer of methionine sulfoxide. However, this activity is not provided by MsrB as expected, but instead by the DMSO reductase complex DmsABC, widely conserved in bacteria. This finding reveals an unexpected diversity in the metabolic enzymes of redox regulation concerning methionine, which should be taken into account in any antibacterial strategies exploiting oxidative stress. DATABASE: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013610.

Function of the evolutionarily conserved plant methionine-S-sulfoxide reductase without the catalytic residue.

In plants, two types of methionine sulfoxide reductase (MSR) exist, namely methionine-S-sulfoxide reductase (MSRA) and methionine-R-sulfoxide reductase (MSRB). These enzymes catalyze the reduction of methionine sulfoxides (MetO) back to methionine (Met) by a catalytic cysteine (Cys) and one or two resolving Cys residues. Interestingly, a group of MSRA encoded by plant genomes does not have a catalytic residue. We asked that if this group of MSRA did not have any function (as fitness), why it was not lost during the evolutionary process. To challenge this question, we analyzed the gene family encoding MSRA in soybean (GmMSRAs). We found seven genes encoding GmMSRAs, which included three segmental duplicated pairs. Among them, a pair of duplicated genes, namely GmMSRA1 and GmMSRA6, was without a catalytic Cys residue. Pseudogenes were ruled out as their transcripts were detected in various tissues and their Ka/Ks ratio indicated a negative selection pressure. In vivo analysis in Δ3MSR yeast strain indicated that the GmMSRA6 did not have activity toward MetO, contrasting to GmMSRA3 which had catalytic Cys and had activity. When exposed to H2O2-induced oxidative stress, GmMSRA6 did not confer any protection to the Δ3MSR yeast strain. Overexpression of GmMSRA6 in Arabidopsis thaliana did not alter the plant's phenotype under physiological conditions. However, the transgenic plants exhibited slightly higher sensitivity toward salinity-induced stress. Taken together, this data suggested that the plant MSRAs without the catalytic Cys are not enzymatically active and their existence may be explained by a role in regulating plant MSR activity via dominant-negative substrate competition mechanism.

Methionine sulfoxide reductase B3 requires resolving cysteine residues for full activity and can act as a stereospecific methionine oxidase.

The oxidation of methionine residues in proteins occurs during oxidative stress and can lead to an alteration in protein function. The enzyme methionine sulfoxide reductase (Msr) reverses this modification. Here, we characterise the mammalian enzyme Msr B3. There are two splice variants of this enzyme that differ only in their N-terminal signal sequence, which directs the protein to either the endoplasmic reticulum (ER) or mitochondria. We demonstrate here that the enzyme can complement a bacterial strain, which is dependent on methionine sulfoxide reduction for growth, that the purified recombinant protein is enzymatically active showing stereospecificity towards R-methionine sulfoxide, and identify the active site and two resolving cysteine residues. The enzyme is efficiently recycled by thioredoxin only in the presence of both resolving cysteine residues. These results show that for this isoform of Msrs, the reduction cycle most likely proceeds through a three-step process. This involves an initial sulfenylation of the active site thiol followed by the formation of an intrachain disulfide with a resolving thiol group and completed by the reduction of this disulfide by a thioredoxin-like protein to regenerate the active site thiol. Interestingly, the enzyme can also act as an oxidase catalysing the stereospecific formation of R-methionine sulfoxide. This result has important implications for the role of this enzyme in the reversible modification of ER and mitochondrial proteins.

Soluble expression of biologically active methionine sulfoxide reductase B1 (PaMsrB1) from Carica papaya in Escherichia coli and isolation of its protein targets.

Plant methionine sulfoxide reductase B1 (MsrB1) protects the photosynthetic apparatus from oxidative damage by scavenging reactive oxygen species to repair Met-oxidized proteins in response to abiotic stresses and biotic attack. Papaya MsrB1 (PaMsrB1) was identified previously to interact with papaya ringspot virus NIa-Pro, and this interaction inhibits the import of PaMsrB1 into the chloroplast. Further functional characterization of PaMsrB1 requires the production of a biologically active purified recombinant protein. In this report, PaMsrB1 as a fusion protein containing an N-terminal maltose-binding protein (MBP) was expressed in Escherichia coli Rosetta (DE3) cells and purified. Production of soluble fusion protein was greater when the cells were cultured at 16 °C than at 37 °C. The Factor Xa protease digested MBP-PaMsrB1 fusion protein and subsequently purified recombinant PaMsrB1 specifically reduced the R-diastereomer of methionine sulfoxide (MetSO) and Dabsyl-MetSO to Met in the presence of dithiothreitol. Eight chloroplast-localized and five non-chloroplast-localized candidate proteins that interact with PaMsrB1 were isolated by affinity chromatography and liquid chromatography coupled to tandem mass spectrometry. The results provide a platform to further understand the anti-oxidative defense mechanism of PaMsrB1.

Functional comparison of methionine sulphoxide reductase A and B in Corynebacterium glutamicum.

Methionine sulphoxide reductases (Msr) are able to reduce methionine sulfoxide to methionine and protect bacteria against reactive oxygen species (ROS). Many organisms express both methionine sulphoxide reductase A (MsrA), specific for methionine-S-sulfoxide and methionine sulphoxide reductase B (MsrB), active against methionine-R-sulfoxide. Corynebacterium glutamicum expresses MsrA, the function of which has been well defined; however, the function of MsrB has not been studied. Whether MsrB and MsrA play an equally important role in the antioxidant process is also poorly understood. In this study, we identified MsrB encoded by ncgl1823 in C. glutamicum, investigated its function and made a comparison with MsrA. The msrB gene showed a slight effect on utilizing methionine sulfoxide (MetO) as the sole Met source; however, the survival rates showed no sensitivity to oxidants. MsrB showed catalytic activity using thioredoxin/thioredoxin reductase (Trx/TrxR) reducing system as electron donors, but independent from the mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/Mtr/MSH) system. Therefore, MsrB plays a limited role in resisting oxidative stress and it could reduce MetO to Met by the Trx/TrxR reducing system, which is useful for expanding the understanding of the functions of Msr in this important industrial microbe.

A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP.

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.

Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.

A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.

A Methionine Residue Promotes Hyperoxidation of the Catalytic Cysteine of Mouse Methionine Sulfoxide Reductase A.

Methionine sulfoxide reductase A (msrA) reduces methionine sulfoxide in proteins back to methionine. Its catalytic cysteine (Cys72-SH) has a low pKa that facilitates oxidation by methionine sulfoxide to cysteine sulfenic acid. If the catalytic cycle proceeds efficiently, the sulfenic acid is reduced back to cysteine at the expense of thioredoxin. However, the sulfenic acid is vulnerable to "irreversible" oxidation to cysteine sulfinic acid that inactivates msrA (hyperoxidation). We observed that human msrA is resistant to hyperoxidation while mouse msrA is readily hyperoxidized by micromolar concentrations of hydrogen peroxide. We investigated the basis of this difference in susceptibility to hyperoxidation and established that it is controlled by the presence or absence of a Met residue in the carboxyl-terminal domain of the enzyme, Met229. This residue is Val in human msrA, and when it was mutated to Met, human msrA became sensitive to hyperoxidation. Conversely, mouse msrA was rendered insensitive to hyperoxidation when Met229 was mutated to Val or one of five other residues. Positioning of the methionine at residue 229 is not critical, as hyperoxidation occurred as long as the methionine was located within the group of 14 carboxyl-terminal residues. The carboxyl domain of msrA is known to be flexible and to have access to the active site, and Met residues are known to form stable, noncovalent bonds with aromatic residues through interaction of the sulfur atom with the aromatic ring. We propose that Met229 forms such a bond with Trp74 at the active site, preventing formation of a protective sulfenylamide with Cys72 sulfenic acid. As a consequence, the sulfenic acid is available for facile, irreversible oxidation to cysteine sulfinic acid.

Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide.

In cells, physiological and pathophysiological conditions may lead to the formation of methionine sulfoxide (MetO). This oxidative modification of methionine exists in the form of two diastereomers, R and S, and may occur in both free amino acid and proteins. MetO is reduced back to methionine by methionine sulfoxide reductases (MSRs). Methionine oxidation was thought to be a nonspecific modification affecting protein functions and methionine availability. However, recent findings suggest that cyclic methionine oxidation and reduction is a posttranslational modification that actively regulates protein function akin to redox regulation by cysteine oxidation and phosphorylation. Methionine oxidation is thus an important mechanism that could play out in various physiological contexts. However, detecting MetO generation and MSR functions remains challenging because of the lack of tools and reagents to detect and quantify this protein modification. We recently developed two genetically encoded diasterospecific fluorescent sensors, MetSOx and MetROx, to dynamically monitor MetO in living cells. Here, we provide a detailed procedure for their use in bacterial and mammalian cells using fluorimetric and fluorescent imaging approaches. This method can be adapted to dynamically monitor methionine oxidation in various cell types and under various conditions.