Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

Abstract

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 Km and kcat were determined to be 24.6 mm and 370 s−1, respectively, for N‐acetyl‐l‐Met at 90 °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 (Zn2+, Mn2+ and Ni2+), and Mn2+ 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.

Genetic and biochemical characterization of arginine biosynthesis in Sinorhizobium meliloti 1021

“…BLASTP analysis of S. meliloti 1021 with Ama1 as the query sequence returned the putative hippurate hydrolases HipO1, HipO2 and SM_b21279 as the only proteins with significant amino acid sequence identity/similarity (30.9-38.2 % to Ama1, as a group). Amino acid residues shown by site-directed mutagenesis to be involved in metal cation-binding and catalysis of a Pyrococcus horikoshii Ama (Tanimoto et al, 2008) are conserved in all four proteins. The three S. meliloti putative hippurate hydrolases share 45.5-49.1 % amino acid identity with one another, but less than 20 % identity with the S. meliloti ArgE.…”

Alteration of metal ions improves the activity and thermostability of aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

“…An active form of wt-PhoACY contained one zinc atom in its subunit whereas a zinc-free form (apoPhoACY) showed no activity, indicating that this metal is essential to express the catalytic function of PhoACY (Tanimoto et al 2008). In the present study, the activities of both wt-and apo-PhoACYs were studied in the presence of some divalent metal ions at a concentration of 1 mM (Table 1).…”

“…The recombinant PhoACY was prepared as described in our previous work (Tanimoto et al 2008). In the present study, the prepared enzyme without any further treatment was denoted as wild-type aminoacylase (wt-PhoACY).…”

“…Measurements of activity and thermostability L-Aminoacylase activity was measured by detecting L-methionine cleaved from N-acetyl-L-methionine (AcMet) as a substrate using the ninhydrin method, according to our previous report (Tanimoto et al 2008). Thermostability of each type of PhoACY was examined by keeping the enzyme in 50 mM Tris/HCl buffer (pH 8.0) at prescribed temperature.…”

“…In our previous study, an L-aminoacylase gene was identified in a hyperthermophilic archeon Pyrococcus horikoshii and its recombinant enzyme (PhoACY) was characterized in terms of the enzymological and kinetic properties (Tanimoto et al 2008). PhoACY was categorized into an M20 metallopeptidase family, similarly to other aminoacylases (Yang et al 1994;Curley et al 2003;JavidMajd and Blanchard 2000;Koreishi et al 2005;Lin et al 2007).…”