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 Pseu domonas fluorescens MFO was cloned and its nucleotide sequence was det ermined. This sequence contains a unique open reading frame (pcp) codi ng for a polypeptide of 213 amino acids (M,22,441) which has significa nt 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 act ive site of these enzymes. The cloned Pcp from P. fluorescens was puri fied to homogeneity and appears to exist as a dimer. This enzyme displ ays a Michaelis constant of 0.21 mM with L-pyroglutamyl-beta-naphthyla mide as the substrate and an absolute substrate specificity towards N- terminal pyroglutamyl residues. Studies of inhibition by chemical comp ounds revealed that the cysteine and histidine residues are essential for enzyme activity. From their conservation in the four enzyme sequen ces, the Cys-144 and His-166 amino acids are proposed to form a part o f the active site of these enzymes.