Molecular cloning and sequence analysis of the X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. cremoris.
Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia coli. A partial XbaI fragment cloned in pUC18 specified X-PDAP activity in E. coli clones. The fragment was also able to confer X-PDAP activity on Bacillus subtilis. The fact that none of these organisms contain this enzymatic activity indicated that the structural gene for X-PDAP had been cloned. The cloned fragment fully restored X-PDAP activity in X-PDAP-deficient mutants of L. lactis. We have sequenced a 3.8-kb fragment that includes the X-PDAP gene and its expression signals. The X-PDAP gene, designated pepXP, comprises 2,289 nucleotide residues encoding a protein of 763 amino acids with a predicted molecular weight of 87,787. No homology was detected between pepXP and genes that had been previously sequenced. A second open reading frame, divergently transcribed, was present in the sequenced fragment; the function or relationship to pepXP of this open reading frame is unknown.
Department of Genetics, University of Groningen, Haren, The Netherlands.
B
B
Mayo
J
J
Kok
K
K
Venema
W
W
Bockelmann
M
M
Teuber
H
H
Reinke
G
G
Venema
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D004269:DNA, Bacterial; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4