Abstract
Mouse, human, and E. coli methionine sulfoxide reductase A (MSRA) stereospecifically catalyze both the reduction of S-methionine sulfoxide to methionine and the oxidation of methionine to S-methionine sulfoxide. Calmodulin has 9 methionine residues, but only Met77 is oxidized by MSRA, and this is completely reversed when MSRA operates in the reductase direction. Given the powerful genetic tools available for Drosophila, we selected this model organism to identify the in vivo calmodulin targets regulated by redox modulation of Met77. The active site sequences of mammalian and Drosophila MSRA are identical, and both contain two cysteine residues in their carboxy terminal domains. We produced recombinant Drosophila MSRA and studied its biochemical and biophysical properties. The enzyme is active as a methionine sulfoxide reductase, but it cannot function as a methionine oxidase. The first step in the mammalian oxidase reaction is formation of a sulfenic acid at the active site, and the second step is the reaction of the sulfenic acid with a carboxy terminal domain cysteine to form a disulfide bond. The third step regenerates the active site through a disulfide exchange reaction with a second carboxy terminal domain cysteine. Drosophila MSRA carries out the first and second steps, but it cannot regenerate the active site in the third step. Thus, unlike the E. coli and mammalian enzymes, Drosophila MSRA catalyzes only the reduction of methionine sulfoxide and not the oxidation of methionine.