FREE METHIONINE-(R)-SULFOXIDE REDUCTASE 2

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. In contrast, fRMsr is a bacterial enzyme specific for the free form of Met-(R)-O. E. coli fRMsr is the first GAF domain family member to show enzymatic activity (Lin et al. 2007 PNAS 104:9597). 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. Moreover, fRMsr may play a key role in maintaining the reduced Met pool for protein synthesis in bacteria. The fRMsr enzyme is an attractive target for drug design as humans do not possess this protein. Site-directed mutagenesis and kinetic analyses have shown that three Cys residues are involved in the fRMsr catalytic mechanism, including a Cys sulfenic acid intermediate. While the mechanism appears to be similar to the other Msr enzymes, the structural basis for the recognition of the substrate is novel. Moreover, two of the Cys residues are located on surface loops that upon MetO reduction result in the formation of a disulfide bond (Cys84-Cys118) and complete enclosure of the active site cavity. Current efforts have lead to the crystallographic determination of the Met product complex. In this complex the active site loops show marked structural rearrangement. The focus of this study is to determine the Cys94-Cys118 reaction intermediate, which should show even more dramatic structural rearrangements.