Selenoprotein R is a zinc-containing stereo-specific methionine sulfoxide reductase

Abstract

Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in the presence of H2O2 and methionine sulfoxide. We found that the cysteine mutant of mouse SelR, as well as the Drosophila SelR homolog, contained zinc and reduced methionine-R-sulfoxide, but not methionine-S-sulfoxide, in in vitro assays, a function that is both distinct and complementary to the stereo-specific activity of MsrA. These findings identify a function of the conserved SelR enzyme family, define a pathway of methionine sulfoxide reduction, reveal a case of convergent evolution of similar function in structurally distinct enzymes, and suggest a previously uncharacterized redox regulatory role of selenium in mammals.

Figure 1

Alignment of SelR homologs. SelR proteins relevant to the studies reported herein are shown in the figure. Sec. structure, secondary structure consensus for the SelR family (generated with jpred2 and sspro). In the secondary structure, H show α-helixes, and E β-strands. A star above the sequence shows the location of conserved selenocysteine (shown as U) and Cys.

Figure 2

Growth of yeast strains in the presence of hydrogen peroxide and methionine sulfoxides. Overnight cultures of wild-type (WT), SelR knockout (SelR), MsrA knockout (MsrA), or SelR/MsrA double knockout (SelR MsrA) yeast strains were diluted to 0.05 OD₆₀₀ in supplemented yeast nitrogen base minimal medium (YNB) and incubated at 30°C with 250 rpm shaking. OD₆₀₀ of the cultures was monitored during the incubation until cells reached stationary phase. Cells were growing in the absence of hydrogen peroxide (A), in 2 mM hydrogen peroxide (B), and in 0.5 mM hydrogen peroxide (C). For utilization of methionine sulfoxides (D), cultures were diluted to 0.2, 0.02, 0.002, and 0.0002 OD₆₀₀ and 10 μl of each cell suspension was applied to spots. Each plate contained 0.7 mM hydrogen peroxide and 0.14 mM of methionine or indicated methionine sulfoxides. Cells were allowed to grow for three days at 30°C, and plates were pictured.

Figure 3

SelR is methionine-R-sulfoxide reductase. (A) Methionine sulfoxide reductase activity of mouse SelR. HPLC assays of reduction of dabsyl L-methionine-S-sulfoxide (upper chromatogram) and L-methionine-R-sulfoxide (lower chromatogram) are shown as detected by absorbance at 436 nm. (B) Methionine sulfoxide reductase activity of Drosophila SelR. HPLC assays of reduction of dabsyl L-methionine-S-sulfoxide (two top chromatograms) and L-methionine-R-sulfoxide (two lower chromatograms) are shown as detected by absorbance at 436 nm. Locations of dabsyl methionine sulfoxide substrates (Met-S-SO and Met-R-SO) and dabsyl methionine product (Met) are indicated. The peak appearing at ≈1.5 min corresponds to dabsyl chloride impurities.

Figure 4

A model for a pathway of methionine sulfoxide reduction. R and S isomers of methionine sulfoxide (chiral centers are located on sulfur as indicated in the figure) are formed directly or indirectly in free and protein methionines in response to oxidants, such as hydrogen peroxide. Oxidation of methionines may modulate activities of proteins, regulate cellular pathways, and disrupt redox homeostasis. Methionine-S-sulfoxides are reduced by MsrA and methionine-R-sulfoxides by SelR with reductants, such as thioredoxin.