Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene.
The structural gene for the Vibrio cholerae leucine aminopeptidase (lap) was cloned and sequenced. The cloned DNA fragment contained a 1,503-bp open reading frame potentially encoding a 501-amino-acid polypeptide with a calculated molecular mass of 54,442 Da. The deduced amino acid sequence of the entire protein showed high homology with the sequence of Vibrio proteolyticus leucine aminopeptidase. The residues potentially involved in binding the zinc ions were completely conserved in the V. cholerae aminopeptidase as well as in the V. proteolyticus aminopeptidase. The recombinant protein was partially purified and characterized. The molecular mass was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 34 kDa, suggesting a processing of the protein to acquire the mature form. The protease showed maximum activity at pH 9.0 and was thermostable at 70 degrees C. The substrate leucyl-p-nitroanilide was cleaved by the protease, and its activity was inhibited by EDTA and bestatin. These results suggested that the protein was a leucine aminopeptidase. The PCR analysis of lap gene distribution showed that it was widely distributed among the V. cholerae strains. It was not present in the other species examined.
Toma
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Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan. k950417@med.u-ryuku.ac.jp.
Honma
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Y
D000595:Amino Acid Sequence; D000813:Anilides / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013696:Temperature; D014734:Vibrio cholerae / Q000201:enzymology / Q000235:genetics*
D000813:Anilides; D011994:Recombinant Proteins; C001536:1-leucine-4-nitroanilide; D007931:Leucyl Aminopeptidase