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N-terminal methionine-specific peptidase in Salmonella typhimurium
Abstract:
Crude extracts of a multiply peptidase-deficient strain of Salmonella typhimurium contain an aminopeptidase that specifically removes N-terminal methionine from peptides. This activity shows pronounced specificity for the peptide's second amino acid. Methionine is removed from peptides with alanine, threonine, or glycine in this position but not when the second amino acid is leucine or methionine. The activity is stimulated by Co2+ and is inhibited by EDTA. Mutations that lead to overproduction (up to 30-fold) of the activity have been obtained by selecting for growth on Met-Gly-Gly as a methionine source. These mutations map at approximately 3 map units, phage P22 cotransducible with leu. The overproducer mutations are dominant to wild type, and duplication of the wild-type allele of the locus leads to a gene dosage effect on peptidase levels. This suggests that the locus of the overproducer mutations may be the structural gene for the peptidase. NaDodSO4/PAGE shows an increased level of a single protein (34 kDa) in the overproducer mutant. This protein is highly enriched in a purified preparation of the peptidase. The specificity of this enzyme suggests that it is involved in the cleavage of methionine from newly synthesized peptide chains. This activity can specifically remove methionine from the N terminus of a completed protein. Treatment of purified, unprocessed (N-terminal methionine) interleukin 1 beta with the purified peptidase results in removal of N-terminal methionine with no additional alterations. N-terminal processing of at least this protein can occur after translation is complete. We propose to call this enzyme peptidase M (methionine-specific aminopeptidase).
Insights
Researchers identified a novel enzyme, peptidase M (methionine-specific aminopeptidase), in Salmonella typhimurium that precisely removes N-terminal methionine from peptides. This discovery offers new insights into post-translational protein modification and processing.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Salmonella typhimurium possesses a multiply peptidase-deficient strain.
- Crude extracts revealed an aminopeptidase activity specifically cleaving N-terminal methionine.
Purpose of the Study:
- To characterize the novel aminopeptidase responsible for N-terminal methionine removal.
- To investigate the enzyme's specificity, regulation, and potential biological role.
Main Methods:
- Enzyme activity assays with various peptide substrates.
- Genetic analysis involving mutation selection and mapping.
- Protein analysis using NaDodSO4/PAGE and purification.
- In vitro processing of interleukin 1 beta.
Main Results:
- The enzyme exhibits specificity for the second amino acid, cleaving methionine when it's Ala, Thr, or Gly, but not Leu or Met.
- Mutations leading to 30-fold overproduction were identified and mapped.
- A 34 kDa protein correlating with peptidase activity was observed.
- The purified enzyme efficiently removed N-terminal methionine from interleukin 1 beta post-translationally.
Conclusions:
- The identified enzyme, named peptidase M, is a methionine-specific aminopeptidase.
- This enzyme plays a role in post-translational processing by removing N-terminal methionine.
- N-terminal methionine cleavage can occur after protein synthesis is complete.