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Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii
Author(s) -
Tanimoto Koichi,
Higashi Noriko,
Nishioka Motomu,
Ishikawa Kazuhiko,
Taya Masahito
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06274.x
Subject(s) - pyrococcus horikoshii , homotetramer , biochemistry , enzyme , amino acid , chemistry , escherichia coli , peptide sequence , stereochemistry , biology , protein subunit , gene
The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii , was cloned and overexpressed in Escherichia coli . The recombinant enzyme was determined to be thermostable aminoacylase ( Pho ACY), forming a homotetramer. Purified Pho ACY showed the ability to release amino acid molecules from the substrates N ‐acetyl‐ l ‐Met, N ‐acetyl‐ l ‐Gln and N ‐acetyl‐ l ‐Leu, but had a lower hydrolytic activity towards N ‐acetyl‐ l ‐Phe. The kinetic parameters K m and k cat were determined to be 24.6 m m and 370 s −1 , respectively, for N ‐acetyl‐ l ‐Met at 90 °C. Purified Pho ACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of Pho ACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn 2+ , Mn 2+ and Ni 2+ ), and Mn 2+ addition caused an alteration in substrate specificity. Site‐directed mutagenesis analysis and structural modeling of Pho ACY, based on Arabidopsis thaliana indole‐3‐acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in Pho ACY, His106, Glu139, Glu140 and His164 were related to the metal‐binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that Pho ACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.