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Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of
S Chevallier1, P Goeltz, P Thibault
1Laboratoire de Biologie Structurale, Commissariat à l'Energie Atomique et Centre National de la Recherche Scientifique, URA 1333, DSV/DBMS, Grenoble, France.
Abstract:
A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.
Insights
Researchers purified a prolyl endopeptidase from Flavobacterium meningosepticum, determining its full protein sequence and identifying the active-site serine. This enzyme shows limited homology to other proteases, highlighting its unique structure.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Prolyl endopeptidases are enzymes that cleave peptide bonds at proline residues.
- Understanding the structure and function of novel proteases is crucial for various biotechnological applications.
Purpose of the Study:
- To purify and characterize a prolyl endopeptidase from Flavobacterium meningosepticum.
- To determine the complete amino acid sequence and identify the active site of the enzyme.
Main Methods:
- Enzyme purification and trypsin digestion.
- Polymerase Chain Reaction (PCR) amplification using specific oligonucleotides.
- Cloning and sequencing of a DNA fragment encoding the prolyl endopeptidase.
- Mass spectrometry for signal peptide identification.
- Active-site labeling with [3H]diisopropyl fluorophosphate.
Main Results:
- A 300-bp fragment was amplified via PCR, leading to the isolation and sequencing of a 2.4-kilobase EcoRI fragment.
- The gene encodes a 705-amino acid protein with an N-terminal signal peptide, resulting in a mature protease of 685 residues (76784 Da).
- Sequence homology was found only with porcine brain prolyl endopeptidase.
- The active-site serine was identified at position 536 within the GRSNGG sequence, distinct from trypsin and subtilisin families.
Conclusions:
- The complete sequence of the Flavobacterium meningosepticum prolyl endopeptidase was elucidated.
- The enzyme possesses a unique active-site sequence, differentiating it from other known serine proteases.
- This characterization provides insights into the structural diversity of prolyl endopeptidases.