Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity

Peptide methionine sulfoxide reductases (MsrA) from many different organism s share a consensus amino acid sequence (GCFWG) that could play an importan t role in their active site. Site-directed single substitution of each of t hese amino acids except glycines in the yeast MsrA resulted in total loss o f enzyme activity, Nevertheless, all the recombinant MsrA mutants and nativ e proteins had a very similar circular dichroism spectrum. The demonstratio n that either treatment with iodoacetamide or replacement of the motif cyst eine with serine leads to inactivation of the enzyme underscores the singul ar importance of cysteine residues in the activity of MsrA. The recombinant yeast MsrA was used for general characterization of the enzyme. Its K-m va lue was similar to the bovine MsrA and appreciably lower than the K-m of th e bacterial enzyme. Also, it was shown that the enzymatic activity increase d dramatically with increasing ionic strength. The recombinant yeast MsrA a ctivity and the reduction activity of free methionine sulfoxide(s) were ste reoselective toward the L-methionine S-sulfoxide and S-methyl p-tolyl sulfo xide. It was established that a methionine auxotroph yeast strain could gro w on either form of L-methionine sulfoxide.