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 organisms share a consensus amino acid sequence (GCFWG) that could play an important role in their active site. Site-directed single substitution of each of these amino acids except glycines in the yeast MsrA resulted in total loss of enzyme activity. Nevertheless, all the recombinant MsrA mutants and native proteins had a very similar circular dichroism spectrum. The demonstration that either treatment with iodoacetamide or replacement of the motif cysteine with serine leads to inactivation of the enzyme underscores the singular importance of cysteine residues in the activity of MsrA. The recombinant yeast MsrA was used for general characterization of the enzyme. Its K(m) value was similar to the bovine MsrA and appreciably lower than the K(m) of the bacterial enzyme. Also, it was shown that the enzymatic activity increased dramatically with increasing ionic strength. The recombinant yeast MsrA activity and the reduction activity of free methionine sulfoxide(s) were stereoselective toward the L-methionine S-sulfoxide and S-methyl p-tolyl sulfoxide. It was established that a methionine auxotroph yeast strain could grow on either form of L-methionine sulfoxide.
Moskovitz
J
J
Laboratory of Biochemistry and Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, Maryland 20982-0320, USA. jmosko@nih.gov
Poston
J M
JM
Berlett
B S
BS
Nosworthy
N J
NJ
Szczepanowski
R
R
Stadtman
E R
ER
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D017931:DNA Primers; D008715:Methionine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D051150:Methionine Sulfoxide Reductases; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D008715:Methionine; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; C013111:methionine sulfoxide