Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene.
The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5 kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110, 315-320].
Yoshimoto
T
T
School of Pharmaceutical Sciences, Nagasaki University.
Kanatani
A
A
Shimoda
T
T
Inaoka
T
T
Kokubo
T
T
Tsuru
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases
D004269:DNA, Bacterial; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases