Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus 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, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Sunaike 2257, Nojicho, Kusatsu, Shiga, 525-0055, Japan. s-tsunas@mx.biwa.or.jp
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Tsunasawa
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Nakura
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Tanigawa
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Kato
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D010450:Endopeptidases; D004795:Enzyme Stability; D004926:Escherichia coli; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008715:Methionine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D004269:DNA, Bacterial; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008715:Methionine; D010450:Endopeptidases; D011751:Pyroglutamyl-Peptidase I