The Potential Active Site of the Lipoprotein-specific (Type II) Signal Peptidase of Bacillus subtilis

Abstract

Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.

An Important Step inListeriaLipoprotein Research

“…Similar to Lgt, the lipoprotein-specific signal peptidase Lsp has been shown to be dispensable for growth in several grampositive bacteria, including L. monocytogenes (6,13,21,23). These findings contrast with those obtained for gram-negative bacteria, in which both Lgt and Lsp are essential enzymes.…”

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

“…1). In contrast to the case for SPI, where the protease interacts reversibly with the membrane, the catalytic residues of SPII appear to be fixed just beneath the membrane surface for signal peptide cleavage from lipoprotein precursors (156). Therefore, the signal peptide cleavage site of these precursors needs to be more exposed to the lipid phase of the membrane, which is probably facilitated by the relatively short H region of lipoprotein signal peptides.…”

“…SPII is an aspartic acid protease with its catalytic residues positioned at the extracytoplasmic membrane surface (156). In B. subtilis SPII, both active site Asp residues 102 and 129 are positioned at the ends of transmembrane segments ( Fig.…”

Maturation of Lipoproteins by Type II Signal Peptidase Is Required for Phagosomal Escape of Listeria monocytogenes

“…In particular, the Asn, Asp, and Ala residues, in domains III and V, which are critical for the activity of B. subtilis SPase II (Ref. 23 and references therein) are conserved (not shown). An lsp transcript was detected by reverse transcriptase-PCR in bacteria grown in broth ("Experimental Procedures"), suggesting that lsp encodes a functional protein.…”