Abstract
Methionine residues in proteins are susceptible to oxidation, and the resulting methionine sulfoxides can be reduced back to methionines by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Herein, we have identified two MsrB families that differ by the presence of zinc. Evolutionary analyses suggested that the zinc-containing MsrB proteins are prototype enzymes and that the metal was lost in certain MsrB proteins later in evolution. Zinc-containing Drosophila MsrB was further characterized. The enzyme was found to employ a catalytic Cys(124) thiolate, which directly interacted with methionine sulfoxide, resulting in methionine and a Cys(124) sulfenic acid intermediate. A subsequent reaction of this intermediate with Cys(69) generated an intramolecular disulfide. Dithiothreitol could reduce either the sulfenic acid or the disulfide, but the disulfide was a preferred substrate for thioredoxin, a natural electron donor. Interestingly, the C69S mutant could complement MsrA/MsrB deficiency in yeast, and the corresponding natural form of mouse MsrB was active with thioredoxin. These data indicate that MsrB proteins employ alternative mechanisms for sulfenic acid reduction. Four other conserved cysteines in Drosophila MsrB (Cys(51), Cys(54), Cys(101), and Cys(104)) were found to coordinate structural zinc. Mutation of any one or a combination of these residues resulted in complete loss of metal and catalytic activity, demonstrating an essential role of zinc in Drosophila MsrB. In contrast, two conserved histidines were important for thioredoxin-dependent activity, but were not involved in zinc binding. A Drosophila MsrA gene was also cloned, and the recombinant enzyme was found to be metal-free and specific for methionine S-sulfoxide and to employ a similar sulfenic acid/disulfide mechanism.
Highlights
The side chains of the sulfur-containing amino acid residues are susceptible to oxidation by reactive oxygen species [1]
Because mouse and Drosophila MsrB proteins catalyze a redox reaction and are zinc-containing proteins [8], we were interested in cysteine residues that could be involved in catalysis as well as in cysteine and histidine residues that could participate in zinc coordination
In contrast to methionine-S-sulfoxide reductase (MsrA), the evolutionary divergence of MsrB proteins resulted in two major families that differ with regard to the mechanism of sulfenic acid reduction and the requirement for structural zinc
Summary
The side chains of the sulfur-containing amino acid residues (cysteine and methionine) are susceptible to oxidation by reactive oxygen species [1]. Such modifications may change protein function, modulate its activity, or result in a signaling event. Selenium is located at enzyme active sites and is involved in redox reactions [11, 12], suggesting that selenocysteine in selenoprotein R and corresponding cysteines in other MsrB homologs are directly involved in catalysis [8]. The structure of MsrB is not known; but the enzyme is predicted to be a -rich protein, and it was suggested that MsrA and MsrB independently evolved their stereo-specific methionine-sulfoxide reductase functions [8]. We report the cloning and characterization of fruit fly MsrA
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