Characterization of the pcp gene of Pseudomonas fluorescens and of its product, pyrrolidone carboxyl peptidase (Pcp).
The gene pcp, encoding pyrrolidone carboxyl peptidase (Pcp), from Pseudomonas fluorescens MFO was cloned and its nucleotide sequence was determined. This sequence contains a unique open reading frame (pcp) coding for a polypeptide of 213 amino acids (M(r) 22,441) which has significant homology to the Pcps from Streptococcus pyogenes, Bacillus subtilis, and Bacillus amyloliquefaciens. Comparison of the four Pcp sequences revealed two highly conserved motifs which may be involved in the active site of these enzymes. The cloned Pcp from P. fluorescens was purified to homogeneity and appears to exist as a dimer. This enzyme displays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthylamide as the substrate and an absolute substrate specificity towards N-terminal pyroglutamyl residues. Studies of inhibition by chemical compounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequences, the Cys-144 and His-166 amino acids are proposed to form a part of the active site of these enzymes.
Gonzales
T
T
Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, U.R.A. Centre National de la Recherche Scientifique, 1486, Institut National des Sciences Appliquées, Villeurbanne, France.
Robert-Baudouy
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics*; D011751:Pyroglutamyl-Peptidase I / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D011751:Pyroglutamyl-Peptidase I