Cloning, nucleotide sequence, and expression of an isovaleryl pepstatin-insensitive carboxyl proteinase gene from Pseudomonas sp. 101.
A unique carboxyl proteinase (EC 3.4.23.33), insensitive to the classical inhibitor isovaleryl pepstatin and isolated from Pseudomonas sp. 101 (PCP), is the first example of a prokaryotic enzyme of this class. The gene coding for PCP was cloned, sequenced, and expressed in Escherichia coli. The gene consists of 1,761 base pairs encoding a protein of 587 amino acid residues. The NH2-terminal 215-amino acid preprosequence flanks the 372-amino acid mature protein, which is identical with the primary structure of an authentic PCP determined by chemical methods. E. coli carrying a plasmid containing the cloned wild-type PCP gene produced a 62-kDa protein. This molecule was processed and secreted into the periplasm as a 43-kDa protein, which converted to mature PCP under acidic conditions. This autocatalytic conversion was completely blocked by tyrostatin, a PCP-specific peptidic inhibitor from Kitasatosporia sp. 55. The purified recombinant PCP has the same characteristics as authentic PCP. When several preprosequence deletion mutants were expressed in E. coli, mutant proteins were accumulated as insoluble forms with no proteinase activities. These results suggest that the prepropeptide of PCP plays an essential role in the formation of functional PCP.
Oda
K
K
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Japan.
Takahashi
T
T
Tokuda
Y
Y
Shibano
Y
Y
Takahashi
S
S
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D010436:Pepstatins; D011994:Recombinant Proteins; C014195:Streptomyces pepsin inhibitor; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin