Sequence of a gene (lap) encoding a 95.3-kDa aminopeptidase from Lactococcus lactis ssp. cremoris Wg2.
A gene (lap) coding for a Lactococcus lactis ssp. cremoris Wg2 aminopeptidase was cloned from genomic libraries of size-fractionated lactococcal DNA. The 5' end of the lap gene was isolated by using a polymerase chain reaction hybridization probe of 77 nucleotides (nt) synthesized from two degenerate primers derived from the N-terminal amino acid (aa) sequence of the lactococcal lysine-aminopeptidase (LAP). The remaining part(s) of the gene were recovered by a search for overlapping sequences in Southern blots of variably restricted genomic DNA. The complete nt sequence of the lap gene has been determined. A large open reading frame of 2538 nt is predicted to encode a polypeptide of 846 aa (approx. 95.3 kDa; pI, 5.93). A recombinant plasmid containing the lap gene with its flanking sequences was shown to direct in vivo synthesis of LAP activity in Escherichia coli, indicating that the cloned DNA fragment is the lap gene. Primer extension analysis of lap mRNA and Northern blot hybridization indicated the gene transcript to be approx. 3.0 kb in size with a 5'-untranslated region of 19-22 nt. Comparison of the deduced aa sequence indicates that the LAP has extensive homology with the super family of Zn(2+)-metallohydrolases and shows identity in the core deca-peptide consensus sequence for the Zn(2+)-binding motif of these enzymes.
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Chr. Hansen's Laboratorium Danmark A/S, Department of Genetics, Hørsholm, Denmark.
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015698:Genomic Library; D006801:Humans; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D000626:Aminopeptidases; C074133:lactococcal lysine aminopeptidase