Free methionine-( R )-sulfoxide reductase from Escherichia coli reveals a new GAF domain function

Abstract

The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S-and R-forms of MetO repaired by MsrA and MsrB, respectively. Cell extracts from an MsrA ؊ B ؊ knockout of Escherichia coli have several remaining Msr activities. This study has identified an enzyme specific for the free form of Met-(R)-O, fRMsr, through proteomic analysis. The recombinant enzyme exhibits the same substrate specificity and is as active as MsrA family members. E. coli fRMsr is, however, 100-to 1,000-fold more active than non-selenocysteine-containing MsrB enzymes for free Met-(R)-O. The crystal structure of E. coli fRMsr was previously determined, but no known function was assigned. Thus, the function of this protein has now been determined. The structural similarity of the E. coli and yeast proteins suggests that most fRMsrs use three cysteine residues for catalysis and the formation of a disulfide bond to enclose a small active site cavity. This latter feature is most likely a key determinant of substrate specificity. Moreover, E. coli fRMsr is the first GAF domain family member to show enzymatic activity. Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms. methionine oxidation ͉ methionine sulfoxide reductase

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

“…Reduced and alkylated peptides were digested overnight at 37°C with trypsin. Peptides were separated by reverse phase chromatography and analyzed directly by electrospray ionization using a nanoelectrospray ionization source on a Bruker Esquire HCT ion trap mass spectrometer (27). MS/MS spectra were searched against the current release of the Mass Spectrometry Protein Sequence Data Base (obtained from the Imperial College London) using Mascot (36).…”

Crystallization and preliminary X-ray crystallographic analysis of free methionine-(R)-sulfoxide reductase fromStaphylococcus aureus

“…To date, only one crystal structure of fRMsr, that from Escherichia coli, is available in the PDB (PDB code 1vhm) and its biochemical function and catalytic mechanism were reported to be unknown (Badger et al, 2005). The X-ray structure suggested that fRMsrs use Cys residues for catalysis and that the active site is enclosed in a small cavity (Lin et al, 2007). The enclosed cavity supports the apparent substrate specificity.…”

“…The fRMsr sequence is conserved in bacteria and yeast but is not found in humans (Lin et al, 2007). The overall folds show that MsrA, MsrB and fRMsr are unrelated.…”

“…Various reactive oxygen and nitrogen species can damage cellular components, including lipids, DNA and proteins (Lin et al, 2007). Damage to proteins is likely to be a consequence of the oxidation of methionine and cysteine residues.…”

“…Recently, an enzyme specific to the reduction of the free form of Met-R-SO has been characterized and named fRMsr (Lin et al, 2007). The fRMsr sequence is conserved in bacteria and yeast but is not found in humans (Lin et al, 2007).…”