Identification of the Amino Acid Residues Essential for Proteolytic Activity in an Archaeal Signal Peptide Peptidase

Abstract

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 (SppA Tk ), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. ⌬N54SppA 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. not only increased activity levels but also broadened the substrate specificity of SppA Tk , 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.

Crystal Structure of a Bacterial Signal Peptide Peptidase

“…5,7,11 The smaller SppA enzymes, which include the characterized Gram-positive Bacillus subtilis SppA BS 5 and the archaeal Thermococcus kodakaraensis SppA TK , 6 are approximately half the size of the larger SppA enzymes and are most homologous to the C-terminal half of SppA EC . Our structure of SppA EC and our sequence analysis (Fig.…”

“…8 Unlike the eukaryotic functional homologue that has been proposed to be an aspartic protease, 9 site-directed mutagenesis along with sequence analysis is consistent with the bacterial, archaeal and thylakoid SppA being a serine protease. 6 …”

Crystal Structure of Bacillus subtilis Signal Peptide Peptidase A

“…In archaea, plant chloroplasts and bacteria, these enzymes [signal peptide peptidase A (SppA)] utilize a serine nucleophile and a lysine general base. [3][4][5][6][7] However, in eukaryotic cells, these enzymes are aspartic proteases. 8 This report focuses on the Gram-positive bacterial serine protease Bacillus subtilis SppA (SppA BS ).…”

Proteolytic inventory of Thermococcus kodakaraensis

“…It belongs to S49 family of serine protease. It is observed that it cleaves the signal peptide on C-terminal of Ala, Leu, Val, Gly and Phe (Matsumi et al, 2006).…”