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.
Highlights
Both cysteine and methionine residues in proteins are susceptible to oxidation by reactive oxygen species [1]
MsrB2 is localised to mitochondria, whereas MsrB3 exists as two main splice variants A and B, containing either a signal sequence targeting the protein to the endoplasmic reticulum (ER) or to the mitochondria, respectively [9]
The protein was expressed in E. coli as a fusion protein with GST which was subsequently cleaved after purification to yield the 21 kDa MsrB3 protein (Figure 1A)
Summary
Both cysteine and methionine residues in proteins are susceptible to oxidation by reactive oxygen species [1]. Characterisation of the role of individual cysteines in the enzymatic process for MsrB3 has suggested that the mechanism of recycling involves the direct reduction of this active site thiol by a thioredoxin domaincontaining protein [11]. A subclass of MsrA enzymes have two resolving cysteines which are thought to act via a threestep process involving disulfide exchange which results in the formation of an internal disulfide bond between the resolving cysteines that is reduced by thioredoxin or low molecular mass thiols [13,14,15] This particular subclass of MsrA enzymes acts as a stereospecific reductase, but has been shown to catalyse the stereospecific oxidation of methionine, a reaction that does not require thioredoxin and results in exclusively the S-epimer [16,17]. We demonstrate that MsrB3 is able to act as an oxidase suggesting a role for methionine oxidation in the regulation of ER protein function
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