Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Rie Matsumi, Haruyuki Atomi, Tadayuki Imanaka

研究成果: Contribution to journalArticle

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Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppATk), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. ΔN54SppA Tk, a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser162 as the nucleophilic serine and that of Lys214 as the general base, comprising a Ser/Lys catalytic dyad in SppATk. Kinetic analyses indicated that Ser184, His191, Lys209, Asp 215, and Arg221 supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser128 and Tyr 165 not only increased activity levels but also broadened the substrate specificity of SppATk, suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.

元の言語英語
ページ(範囲)10533-10539
ページ数7
ジャーナルJournal of Biological Chemistry
281
発行部数15
DOI
出版物ステータス出版済み - 4 14 2006

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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