Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme.
A strain of Aeromonas hydrophila was found to show prolyl endopeptidase activity. The enzyme gene was cloned and expressed in Escherichia coli JM83. A 12 kbp EcoRI fragment containing the enzyme gene was subcloned at the HincII site of pUC19 to construct plasmid pAPEP-3 with a 3.5 kbp insert. E. coli JM83 transformed with this plasmid showed about 100-fold higher activity than the parent Aeromonas. Analysis of the nucleotide sequence of the insert revealed that the mature enzyme-encoding sequence starts just after the ATG initiation codon of the open reading frame. The enzyme was a single polypeptide composed of 689 amino acid residues with a molecular weight of 76,383. It showed properties very similar to those of Flavobacterium prolyl endopeptidase, except that the isoelectric point was 5.5. The amino acid sequence was 56 and 41% homologous to those of Flavobacterium and porcine brain prolyl endopeptidases, respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-537, His-656, and Asp-512 (or Asp-621).
Kanatani
A
A
School of Pharmaceutical Sciences, Nagasaki University.
Yoshimoto
T
T
Kitazono
A
A
Kokubo
T
T
Tsuru
D
D
D016980:Aeromonas hydrophila / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics*; D013045:Species Specificity
D004269:DNA, Bacterial; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases