New deblocking aminopeptidases from Pyrococcus horikoshii.
It has been reported that one of the hyperthermostable aminopeptidases from Pyrococcus horikoshii exhibits hydrolytic activity toward short peptides and acyl-peptides (deblocking activity). In the genome database of P. horikoshii, two new open reading frames homologous to the hyperthermostable aminopeptidase of P. horikoshii were found. The two new genes for the proteins were cloned, expressed using E. coli, and characterized. The purified proteins gave a single band on SDS-PAGE corresponding to molecular masses of 42 kDa and 41 kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. These enzymes are likely to exist as oligomeric structures at neutral pH. The optimum pHs of the two enzyme activities were in the range of 7.0 to 7.5, and the optimum temperatures for the activities were around 100 degrees C. The enzymes exhibited low hydrolytic activity for peptide substrates longer than 10 residues. They were activated by cobalt and zinc ions. Their substrate specificities and activation factors are different. It was confirmed that P. horikoshii has three similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small peptides in P. horikoshii cells.
Mori
Kazushige
K
National Institute of Advanced Industrial Science and Technology (AIST Kansai), Ikeda, Osaka, Japan. kazu-ishikawa@aist.go.jp
Ishikawa
Kazuhiko
K
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D019843:Archaeal Proteins; D009842:Oligopeptides; D000626:Aminopeptidases