PYRROLIDONE CARBOXYL PEPTIDASE FROM THE HYPERTHERMOPHILIC ARCHAEON PROCOCCUS-FURIOSUS - CLONING AND OVEREXPRESSION IN ESCHERICHIA-COLI OF THE GENE, AND ITS APPLICATION TO PROTEIN-SEQUENCE ANALYSIS

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglu tamyl peptidase), which removes amino-terminal pyroglutamyl residues f rom peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenc ed, and expressed in Escherichia coli. The DNA sequence encodes a prot ein containing 208 amino acid residues with methionine at the N-termin us, Analysis of the recombinant protein expressed in E. coli, includin g amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides g enerated by enzymatic digestions with lysylendopeptidase and Staphyloc occus aureus V8 protease, showed its primary structure to be completel y identical with that deduced from its cDNA sequence. Comparison of th e amino acid sequence of P, furiosus Pcp (P.f.Pcp) with those of bacte rial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the cataly tic triad, Cys142, His166, and Glu79, On the other hand, a unique shor t stretch sequence (positions around 175-185) that is absent in bacter ial Pcps was found in P,f,Pcp, A similar stretch has also been reporte d recently in the amino acid sequence of Pcp from the hyperthermophili c Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the ''International Congress on Exthermophiles '98'' p, 58 (1998)], T o elucidate their contribution to the hyperthermostability of these en zymes, further structural studies are required.