| Methionine sulfoxide reductases (Msr) reduce methionine sulfoxide (Met-(O)) back to methionine (Met). This process is stereospecific with the S- and R-forms of Met-(O) repaired by MsrA and MsrB, respectively. In this study, we identified fRMsr from a cell extract of E. coli MsrA-B- double knockout strain through proteomic analysis. As compared to MsrA and MsrB, fRMsr is only specific for the free form of Met-R-(O). The structure of E. coli fRMsr shows that it has a restrictive active site and conserved residues, which explained its substrate specificity. Moreover, E. coli fRMsr belongs to the GAF family of signal transduction domains, and is the first GAF domain shown to exhibit catalytic activity. In order to explore the catalytic mechanism of fRMsr, a series of site-directed mutants of conserved residues were made. The results show that the catalytic process of fRMsr GAF involves the formation of sulfenic acid intermediate and a disulfide bond shift. The structure of the product-enzyme complex of bovine MsrA (bMsrA) was also solved to 1.6 A resolution. The binding mode of the Met residue to the exposed active site of bMsrA, together with kinetic data, explain the broad spectrum of substrates of bMsrA. The enclosed active site of E. coli fRMsr is in stark contrast to the exposed active site of bMsrA. |
