Thermostable aminopeptidase from Pyrococcus horikoshii

From the genome sequence data of the thermophilic archaeon Pyrococcus horik oshii, an open reading frame was found which encodes a protein (332 amino a cids) homologous with an endoglucanase from Clostridium thermocellum (42% i dentity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gen e was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was no t detected, this protein was found to have aminopeptidase activity to cleav e the N-terminal amino acid from a variety of substrates including both N-b locked and non-blocked peptides, The enzyme was stable at 90 degrees C, wit h the optimum temperature over 90 degrees C. The metal ion bound to this en zyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N-alpha-acylated residues in short peptide sequence analysis at high temperatures. (C) 1999 Federation of Eur opean Biochemical Societies.