Acylamino Acid-releasing Enzyme from the Thermophilic ArchaeonPyrococcus horikoshii

Abstract

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°C, with its optimum temperature over 90°C. 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 N ␣ -acylated residues in short peptide sequence analysis at high temperatures.The acylamino acid-releasing enzyme (AARE) 1 catalyzes the NH 2 -terminal hydrolysis of N ␣

Thermostable aminopeptidase from Pyrococcus horikoshii

“…The ampli¢ed gene was expressed using the pET11a vector system in the host E. coli BL21(DE3) according to the instructions by the manufacturer. The host E. coli BL21(DE3) was ¢rst transformed using the constructed plasmid, after which the production of the protein was performed according to the method described previously [2]. The concentration of the expressed protein was determined using a Coomassie protein assay reagent (Pierce Chemical Company, Rockford, IL, USA) and utilizing bovine serum albumin as the standard protein.…”

“…The gene was ampli¢ed using the polymerase chain reaction (PCR) with primers having NdeI and Bam-HI restriction sites according to the method reported previously [2]. The sequences of the primers were: 5P-TTTTGAATTCTTGCATAT-GATGTCAATATAGAGAAG-3P (upper primer, containing an NdeI cutting site as underlined) and 5P-TTTTGGTACCTTTGGATCCT-TATCCCTCCTAGAGCTCAAATGCTAA (lower primer, containing a BamHI cutting site as underlined).…”

“…The acylamino acid releasing enzyme from this organism was shown to be highly thermophilic [2] and the other enzymes are also expected to be highly thermophilic. The sequencing of the genome of this organism was completed at the National Institute of Technology and Evaluation (Tokyo, Japan) [3,4].…”

Expression, purification, crystallization and preliminary crystallographic analysis of a deblocking aminopeptidase fromPyrococcus horikoshii

“…Pyrococcus horikoshii OT3 is a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the Okinawa trough (Gonzalez et al, 1998), the complete genome of which has been sequenced (Kawarabayasi et al, 1998). This archaeon is able to grow at temperatures of between 361 and 377 K, with an optimal growth temperature of 371 K. The enzymes produced by this hyperthermophilic organism are extremely heat-resistant and may therefore be utilized in various industrial ®elds such as depollution, food, biomedical engineering etc.…”

“…Very few proteases from hyperthermophilic microorganisms have been characterized biochemically so far and proteolysis in these organisms is poorly understood despite the availability of complete genome sequences (Ward et al, 2002). Only three other peptidases from P. horikoshii have been studied (Du et al, 2000;Ishikawa et al, 1998;Sokabe et al, 2002). We have therefore undertaken a structural study of DAP in order to better understand its proteolytic function.…”