Resolving oxidative damage to methionine by an unexpected membrane-associated stereoselective reductase discovered using chiral fluorescent probes
Non-enzymatic oxidative processes in living organisms are one of the inevitable consequences of respiration and environmental conditions. They 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 functional consequences. Methionine oxidation can be reverted enzymatically by methionine sulfoxide reductases (Msrs). The two enzyme classes known to fulfil this role are MsrA (reducing (S)-sulfoxides), and MsrB (reducing (R)-sulfoxides). They are strictly stereoselective and conserved throughout the tree of life. Under stress conditions such as stationary phase and nutrient starvation, E.coli upregulates expression of MsrA but similar effect has not been described for MsrB, raising a conundrum of the pathway enabling 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, (R)-sulfoxide-reducing counterpart. 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.
Sample Processing Protocol
Cell debris pellets in 100 mM triethylammonium bicarbonate (TEAB) buffer containing 1% sodium deoxycholate (SDC) were boiled at 95°C for 5 min. Protein concentration was determined using BCA protein assay kit (Thermo) and 20 µg of protein per sample was used for MS sample preparation. Cysteines were reduced with 5 mM final concentration of tris(2-carboxyethyl)phosphine (TCEP) at 60°C for 60 min, and blocked with 10 mM S-methyl methanethiosulfonate (MMTS) for 10 min at room temperature. Samples were digested with trypsin (trypsin/protein ratio of 1/20) at 37°C overnight. After digestion samples were acidified with trifluoroacetic acid (TFA) to 1% final concentration. SDC was removed by extraction to ethylacetate and peptides were desalted on Michrom C18 colum . Nano reversed phase column (EASY-Spray column, 50 cm × 75 µm ID, PepMap C18, 2 µm particles, 100 Å pore size) was used for LC/MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. Samples were loaded onto the trap column (Acclaim PepMap300, C18, 5 µm, 300 Å Wide Pore, 300 µm × 5 mm) at a flow rate of 15 μl/min. Loading buffer was composed of water, 2% acetonitrile and 0.1% TFA. Peptides were eluted with gradient of B from 4% to 35% over 60 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT- qIT, Thermo). Survey scans of peptide precursors from 400 to 1600 m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.5 Da with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104, and the max injection time was 35 ms. Only those precursors with charge state 2–6 were sampled for MS2. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2 s cycles .
Data Processing Protocol
All data were analyzed and quantified with the MaxQuant software (version 126.96.36.199) . The false discovery rate (FDR) was set to 1% for both proteins and peptides and we specified a minimum length of seven amino acids. The Andromeda search engine was used for the MS/MS spectra search against the Escherichia coli database (downloaded from Uniprot on November 2016, containing 6 072 entries). Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Dithiomethylation of cysteine was selected as fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications. The “match between runs” feature of MaxQuant was used to transfer identifications to other LC-MS/MS runs based on their masses and retention times (maximum deviation 0.7 min) and this was also used in quantification experiments. Quantifications were performed with the label-free algorithms described recently . Data analysis was performed using Perseus 188.8.131.52 software . Missing values were imputed from random distribution around the value of instrument sensitivity limit after filtering out contaminants and reverse hits. The imputed data (Supplementary table 1) were plotted into a volcano plot (Fig. 4A) with false discovery rate (FDR) of 0.05 estimated by a permutation-based approach using the S parameter of 0.1 and 250 permutations.
Makukhin N, Havelka V, Poláchová E, Rampírová P, Tarallo V, Strisovsky K, Míšek J. Resolving oxidative damage to methionine by an unexpected membrane-associated stereoselective reductase discovered using chiral fluorescent probes. FEBS J. 2019 PubMed: 31166082