Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.
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.
Chevallier
S
S
Laboratoire de Biologie Structurale, Commissariat à l'Energie Atomique et Centre National de la Recherche Scientifique, URA 1333, DSV/DBMS, Grenoble, France.
Goeltz
P
P
Thibault
P
P
Banville
D
D
Gagnon
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology*; D007531:Isoflurophate / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D000085822:Prolyl Oligopeptidases; D021382:Protein Sorting Signals / Q000302:isolation & purification; D015183:Restriction Mapping; D016415:Sequence Alignment; D012697:Serine Endopeptidases; D014357:Trypsin / Q000378:metabolism
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D007531:Isoflurophate; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D014357:Trypsin