Abstract
The oxidation of methionine residues in proteins occurs during oxidative stress and can lead to an alteration in protein function. The enzyme methionine sulfoxide reductase (Msr) reverses this modification. Here, we characterise the mammalian enzyme Msr B3. There are two splice variants of this enzyme that differ only in their N-terminal signal sequence, which directs the protein to either the endoplasmic reticulum (ER) or mitochondria. We demonstrate here that the enzyme can complement a bacterial strain, which is dependent on methionine sulfoxide reduction for growth, that the purified recombinant protein is enzymatically active showing stereospecificity towards R-methionine sulfoxide, and identify the active site and two resolving cysteine residues. The enzyme is efficiently recycled by thioredoxin only in the presence of both resolving cysteine residues. These results show that for this isoform of Msrs, the reduction cycle most likely proceeds through a three-step process. This involves an initial sulfenylation of the active site thiol followed by the formation of an intrachain disulfide with a resolving thiol group and completed by the reduction of this disulfide by a thioredoxin-like protein to regenerate the active site thiol. Interestingly, the enzyme can also act as an oxidase catalysing the stereospecific formation of R-methionine sulfoxide. This result has important implications for the role of this enzyme in the reversible modification of ER and mitochondrial proteins.