We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KODI. SppATk, 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 (AN54SppATk) was found to be stable against autoproteolysis and was examined further. AN54SppATk 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.ΔN54SppATk displayed a Km of 240 ± 18 ̀M and a Vmax 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, AN54SppATk 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 ΔN54SppATk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.
All Science Journal Classification (ASJC) codes
- Molecular Biology