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Journal of Bacteriology, October 2005, p. 7072-7080, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7072-7080.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Received 30 April 2005/ Accepted 27 July 2005

We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppA_Tk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppA_Tk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (N54SppA_Tk) was found to be stable against autoproteolysis and was examined further. N54SppA_Tk exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. N54SppA_Tk displayed a K_m of 240 ± 18 µM and a V_max of 27.8 ± 0.7 µmol min^–1 mg^–1 towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80°C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, N54SppA_Tk was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of N54SppA_Tk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.

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