Catalytic advantages provided by selenocysteine in methionine-S-sulfoxide reductases

Abstract

Methionine sulfoxide reductases are key enzymes that repair oxidatively damaged proteins. Two distinct stereospecific enzyme families are responsible for this function: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In the present study, we identified multiple selenoprotein MsrA sequences in organisms from bacteria to animals. We characterized the selenocysteine (Sec)-containing Chlamydomonas MsrA and found that this protein exhibited 10-50-fold higher activity than either its cysteine (Cys) mutant form or the natural mouse Cys-containing MsrA, making this selenoenzyme the most efficient MsrA known. We also generated a selenoprotein form of mouse MsrA and found that the presence of Sec increased the activity of this enzyme when a resolving Cys was mutated in the protein. These data suggest that the presence of Sec improves the reduction of methionine sulfoxide by MsrAs. However, the oxidized selenoprotein could not always be efficiently reduced to regenerate the active enzyme. Overall, this study demonstrates that sporadically evolved Sec-containing forms of methionine sulfoxide reductases reflect catalytic advantages provided by Sec in these and likely other thiol-dependent oxidoreductases.

Figure 1. Identification of Sec-containing MsrAs

(A) Multiple sequence alignment of MsrAs. Sec (U) residues are highlighted in red and the corresponding Cys residues are highlighted in blue. The conserved resolving Cys in C-terminal regions of Cys-containing MsrAs are shown in green. Cys55 and Cys95 of C. reinhardtii MsrA are highlighted in yellow. Accession numbers are indicated on the left. Only partial A. variegatum sequence is available. (B) Representative SECIS elements from S. fumaroxidans (bacterium, left), B. microplus (invertebrate animal, middle), and C. reinhardtii (green algae, right) selenoprotein MsrAs. Functionally important nucleotides in SECIS elements are highlighted in red. Sec codon (UGA) upstream of the bacterial SECIS element is highlighted in blue. Eukaryotic SECIS element structures were predicted with SECISearch (5) and the bacterial SECIS was predicted with RNAfold (20). (C) Phylogenetic analysis of Sec- (red) and Cys-containing (black) MsrAs.

Figure 1. Identification of Sec-containing MsrAs

(A) Multiple sequence alignment of MsrAs. Sec (U) residues are highlighted in red and the corresponding Cys residues are highlighted in blue. The conserved resolving Cys in C-terminal regions of Cys-containing MsrAs are shown in green. Cys55 and Cys95 of C. reinhardtii MsrA are highlighted in yellow. Accession numbers are indicated on the left. Only partial A. variegatum sequence is available. (B) Representative SECIS elements from S. fumaroxidans (bacterium, left), B. microplus (invertebrate animal, middle), and C. reinhardtii (green algae, right) selenoprotein MsrAs. Functionally important nucleotides in SECIS elements are highlighted in red. Sec codon (UGA) upstream of the bacterial SECIS element is highlighted in blue. Eukaryotic SECIS element structures were predicted with SECISearch (5) and the bacterial SECIS was predicted with RNAfold (20). (C) Phylogenetic analysis of Sec- (red) and Cys-containing (black) MsrAs.

Figure 2. ⁷⁵Se metabolic labeling of NIH 3T3 cells expressing GST-fused Chlamydomonas MsrA (A) and GST-fusion selenoprotein form of mouse MsrA (B)

The cells were transfected with an empty vector (lane 1) or GST-fusion constructs containing selenoprotein MsrAs (lane 2). The transfected cells were incubated for 24 h in the medium containing ⁷⁵Se-selenite. The proteins were subjected to SDS-PAGE and the labeled proteins were visualized with a PhosphorImager. Endogenous selenoproteins thioredoxin reductase 1 (TR1, 55 kDa) and glutathione peroxidase 1 (GPx1, 25 kDa) are indicated on the left and the selenoproteins expressed from transfected constructs on the right.

Figure 3. Schematic representation of MsrA sequences and location of resolving Cys

Catalytic Cys or Sec (U) are indicated by an arrow. A resolving Cys that forms a disulfide bond with the catalytic Cys is indicated by a dashed arrow. Cys228 of mouse MsrA and Cys207 of E. coli MsrA are involved in the regeneration of the active enzymes by forming a disulfide with the resolving Cys.