Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii.
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 (PhoACY), forming a homotetramer. Purified PhoACY 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 mm and 370 s(-1), respectively, for N-acetyl-l-Met at 90 degrees C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY 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 PhoACY, based on Arabidopsis thaliana indole-3-acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, 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 PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.
Tanimoto
Koichi
K
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka, Japan.
Higashi
Noriko
N
Nishioka
Motomu
M
Ishikawa
Kazuhiko
K
Taya
Masahito
M
D000581:Amidohydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D006358:Hot Temperature; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D019843:Archaeal Proteins; D008670:Metals; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019672:aminoacylase I