ACYLAMINO ACID-RELEASING ENZYME FROM THE THERMOPHILIC ARCHAEON PYROCOCCUS-HORIKOSHII

When the genome of the thermophilic archaeon Pyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino a cid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's pro posed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codo n and a TAG termination codon, encoding a 72,390-Da protein of 632 ami no acid residues. This gene was overexpressed in Escherichia coli with the pET vector system, and the resulting enzyme showed the anticipate d amino-terminal sequence and high hydrolytic activity for acylpeptide s. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degrada tion or initiation of protein synthesis or both in archaea may be simi lar to those in eukaryotes. The enzyme was stable at 90 degrees C, wit h its Optimum temperature over 90 degrees C. The specific activity of the enzyme increased 7-14-fold with heat treatment, suggesting the mod ification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of N-al pha-acylated residues in short peptide sequence analysis at high tempe ratures.