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 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 (deltaN54SppA(Tk)) was found to be stable against autoproteolysis and was examined further. DeltaN54SppA(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. DeltaN54SppA(Tk) displayed a K(m) of 240 +/- 18 microM and a V(max) of 27.8 +/- 0.7 micromol min(-1) mg(-1) towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80 degrees 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, deltaN54SppA(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 deltaN54SppA(Tk) was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.
Matsumi
Rie
R
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Atomi
Haruyuki
H
Imanaka
Tadayuki
T
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics*
D019843:Archaeal Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase