Isolation of the leucine aminopeptidase gene from Aeromonas proteolytica. Evidence for an enzyme precursor

The leucine aminopeptidase of Aeromonas proteolytica (EC 3.4.11.10) is a monomeric metalloenzyme having the capacity to bind two Zn2+ atoms in the active site. Structural information of this relatively small aminopeptidase that could illuminate the catalytic mechanism of the metal ions is lacking; h...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-04, Vol.267 (12), p.8390-8395
Main Authors: GUENET, C, LEPAGE, P, HARRIS, BA
Format: Article
Language:English
Subjects:
Aeromonas - enzymology
Aeromonas proteolytica
Amino Acid Sequence
Base Sequence
Biochemistry & Molecular Biology
Biological and medical sciences
Blotting, Western
cytosol aminopeptidase
DNA, Bacterial - genetics
Electrophoresis, Polyacrylamide Gel
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gene Expression
genes
Genes. Genome
Leucyl Aminopeptidase - genetics
Leucyl Aminopeptidase - metabolism
Life Sciences & Biomedicine
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Nucleic Acid Hybridization
nucleotide sequence
predictions
Protein Sorting Signals - genetics
Protein Sorting Signals - metabolism
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Science & Technology
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Summary:The leucine aminopeptidase of Aeromonas proteolytica (EC 3.4.11.10) is a monomeric metalloenzyme having the capacity to bind two Zn2+ atoms in the active site. Structural information of this relatively small aminopeptidase that could illuminate the catalytic mechanism of the metal ions is lacking; hence, we have obtained sequences from the purified enzyme, cloned the corresponding gene, and expressed the recombinant protein in Escherichia coli. The deduced primary amino acid sequence of this secreted protease suggests a potential signal peptide at the NH2 terminus. Expression of the recombinant and native proteins in E. coli and in extracts of culture media of A. proteolytica indicates that the aminopeptidase is secreted as an active and thermosensitive 43-kDa protein that is rapidly transformed to thermostable forms of 30 and 32 kDa. Comparison of the deduced amino acid sequence of the A. proteolytica leucine aminopeptidase with other Zn(2+)-binding metalloenzymes failed to show homologies to the consensus binding sequence His-Glu-X-X-His for the metal ion.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)42457-x