LAUSR

Methionine sulfoxide reductase A (MsrA) stereospecifically catalyzes the reduction of S-methionine sulfoxide to methionine and is important in defense against oxidative stress. Recently, we reported that mammalian methionine sulfoxide reductase A stereospecifically and selectively oxidizes Met77 in calcium-bound calmodulin and can fully reduce it as well. The control mechanism that prevents futile cycling is hypothesized to be through interaction with a postulated regulatory protein. Thus, cyclic oxidation and reduction of methionines in proteins by MsrA could function as a redox-based mechanism of cellular regulation. Our aim in this study was to elucidate the physiological significance of methionine sulfoxide reductase A mediated reversible oxidation of calmodulin Met77 in Drosophila. However, we found that Drosophila MsrA, unlike its mammalian counterpart, is not a methionine oxidase. This led us to explore the mechanistic details of the enzyme. Using a double alkylation approach with HPLC-mass spectrometric sequencing, we found that the active site cysteine residue in Drosophila MsrA becomes locked in a disulfide bond with the terminal cysteine residue of the protein and thus cannot mediate oxidation. A mutant Drosophila MsrA lacking the two C-terminal cysteine residues also lacked oxidase activity, despite not being able to form a disulfide bond with the active site cysteine.