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 acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coli with the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. 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 degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 degreesC, with its optimum temperature over 90 degreesC. The specific activity of the enzyme increased 7-14-fold with heat treatment, suggesting the modification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of Nalpha-acylated residues in short peptide sequence analysis at high temperatures.
Ishikawa
K
K
National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan. ishikawa@nibh.go.jp
Ishida
H
H
Koyama
Y
Y
Kawarabayasi
Y
Y
Kawahara
J
J
Matsui
E
E
Matsui
I
I
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C038220:acylaminoacyl-peptidase