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Purification and characterization of the Streptococcus salivarius methionine aminopeptidase (MetAP)
El Houssine Boufous1, Christian Vadeboncoeur
1Groupe de recherche en écologie buccale, Département de biochimie et de microbiologie, Faculté des sciences et de génie, and Faculté de médecine dentaire, Université Laval, Quebec, Canada G1K 7P4.
Abstract:
Streptococcus salivarius methionine aminopeptidase (MetAP) was purified from a recombinant Escherichia coli strain containing the S. salivarius map gene, which codes for MetAP. S. salivarius map coded for a protein of 286 amino acids with a calculated molecular mass of 31,723 Da and a pI of 4.6. The native enzyme eluted from a Superdex column as a protein with a molecular mass of 30.6 kDa and cleaved N-terminal Met of peptide only when the penultimate amino acid was Gly, Ala, Ser, Val, Pro, or Thr. The enzyme was more active against tetrapeptides than tripeptides and did not recognize dipeptides. It required the presence of a metal cation for activity, with a preference for Co(2+) over Mn(2+). S. salivarius MetAP has a pH optimum of 8.0 and an optimal temperature at 50 degrees C. The S. salivarius protein had an extra sequence of 24 amino acids between two conserved aspartate residues involved in the coordination of the metal ion. A similar extra sequence is present in MetAP from other streptococci and from Lactococcus lactis, but not from other bacteria or eukaryotes.
Insights
Streptococcus salivarius methionine aminopeptidase (MetAP) selectively cleaves N-terminal methionine from peptides, preferring specific amino acids and requiring metal ions like cobalt for activity.
Area of Science:
- Biochemistry
- Enzymology
- Microbial protein analysis
Background:
- Methionine aminopeptidases (MetAPs) are crucial enzymes involved in protein processing.
- Understanding MetAP specificity is vital for fields like drug discovery and biotechnology.
- The MetAP from Streptococcus salivarius has not been extensively characterized.
Purpose of the Study:
- To purify and characterize the Streptococcus salivarius methionine aminopeptidase (MetAP).
- To determine the substrate specificity and optimal conditions for S. salivarius MetAP activity.
Main Methods:
- Recombinant expression of the S. salivarius map gene in Escherichia coli.
- Purification of the enzyme using Superdex chromatography.
- Enzyme activity assays with various peptide substrates and metal ions.
- Determination of optimal pH and temperature.
Main Results:
- Purified S. salivarius MetAP is a 30.6 kDa protein.
- The enzyme specifically cleaves N-terminal methionine when the penultimate amino acid is Gly, Ala, Ser, Val, Pro, or Thr.
- Optimal activity was observed at pH 8.0 and 50°C, with a preference for Co(2+).
- A unique 24-amino acid insertion was identified in the metal-binding region, present in other streptococci and Lactococcus lactis MetAPs.
Conclusions:
- S. salivarius MetAP exhibits distinct substrate specificity and metal ion preference.
- The enzyme's unique structural feature may influence its catalytic activity and evolutionary relationships.
- This characterization provides insights into bacterial MetAP function and diversity.
