Biochemical properties of a putative signal peptide peptidase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppA_Tk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KODI. 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 (AN54SppA_Tk) was found to be stable against autoproteolysis and was examined further. AN54SppA_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.ΔN54SppA_Tk displayed a K_m of 240 ± 18 ̀M 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, AN54SppA_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 ΔN54SppA_Tk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.