Overexpression and Characterization of a Carboxypeptidase from the Hyperthermophilic Archaeon Thermococcus sp. NA1
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Genomic analysis of a hyperthermophilic archaeon, <I>Thermococcus</I> sp. NA1, revealed the presence of an 1,497 bp open reading frame, encoding a protein of 499 amino acids. The deduced amino acid sequence was similar to thermostable carboxypeptidase 1 from <I>Pyrococcus furiosus</I>, a member of peptidase family M32. Five motifs, including the HEXXH motif with two histidines coordinated with the active site metal, were conserved. The carboxypeptidase gene was cloned and overexpressed in <I>Escherichia coli</I>. Molecular masses assessed by SDS–PAGE and gel filtration were 61 kDa and 125 kDa respectively, which points to a dimeric structure for the recombinant enzyme, designated TNA1_CP. The enzyme showed optimum activity toward Z-Ala-Arg at pH 6.5 and 70–80 °C (<I>k</I><SUB>cat</SUB>⁄<I>K</I><SUB>m</SUB>=8.3 m<small>M</small><SUP>−1</SUP> s<SUP>−1</SUP>). In comparison with that of <I>P. furiosus</I> CP (<I>k</I><SUB>cat</SUB>⁄<I>K</I><SUB>m</SUB>=667 m<small>M</small><SUP>−1</SUP> s<SUP>−1</SUP>), TNA1_CP exhibited 80-fold lower catalytic efficiency. The enzyme showed broad substrate specificity with a preference for basic, aliphatic, and aromatic C-terminal amino acids. This broad specificity was confirmed by C-terminal ladder sequencing of porcine <I>N</I>-acetyl-renin substrate by TNA1_CP.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(5), 1140-1147, 2006-05-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry