Biochemical characterization of deblocking aminopeptidase from hyperthermophilic Archaeon Thermococcus onnurineus NA1

A genomic analysis of the hyperthermophilic archaeon Thermoccoccus onnurineus NA1 (TNA1) revealed the presence of a deblocking aminopeptidase (DAP) gene with high similarity to the genes of DAPs from Pyrococcus furiosus (86%) and Pyrococcus horikoshii (83% identity). The optimum aminopeptidase activity of the recombinant enzyme was observed at pH 7.5 and in the range of 90 degrees C to 100 degrees C. The specific aminopeptidase and deblocking activities of the enzyme toward Leu-pNA and Ac-Leu-pNA were 18- and 3-fold higher than those of a P horikoshii DAP (DAP2), respectively. The enzyme activity was significantly increased by Co2+ ions. The presence of Co2+ ions induced the activation of the enzyme with heating and changed the large oligomer to a dimer. The enzyme activated by Co2+ ions appeared to eventually be inactivated by autodegradation, which was confirmed by mass spectrometry.