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Biochemistry Biochemistry Z 0250 Signal Peptidases -[314 refs.]. -(PAETZEL, M.; KARLA, A.; STRYNADKA, N. C. J.; DALBEY*, R. E.; Chem. Rev. (Washington, D. C.) 102 (2002) 12, 4549-4579; Dep. Chem., Ohio State Univ., Columbus, OH 43210, USA; Eng.) -Lindner 06-271

Type I signal peptidase (SPase I) catalyzes the cleavage of the amino-terminal signal sequences from preproteins destined for cell export. Preproteins contain a signal sequence with a positively charged n-region, a hydrophobic h-region, and a neutral but polar c-region. Despite having no distinct consensus sequence other than a commonly found c-region "Ala-X-Ala" motif preceding the cleavage site, signal sequences are recognized by SPase I with high fidelity. Remarkably, other potential Ala-X-Ala sites are not cleaved within the preprotein. One hypothesis is that the source of this fidelity is due to the anchoring of both the SPase I enzyme (by way of its transmembrane segment) and the preprotein substrate (by the h-region in the signal sequence) in the membrane. This limits the enzyme-substrate interactions such that cleavage occurs at only one site. In this work we have, for the first time, successfully isolated Bacillus subtilis type I signal peptidase (SipS) and a truncated version lacking the transmembrane domain (SipS-P2). With purified full-length as well as truncated constructs of both B. subtilis and Escherichia coli (Lep) SPase I, in vitro specificity studies indicate that the transmembrane domains of either enzyme are not important determinants of in vitro cleavage fidelity, since enzyme constructs lacking them reveal no alternate site processing of pro-OmpA nuclease A substrate. In addition, experiments with mutant pro-OmpA nuclease A substrate constructs indicate that the h-region of the signal peptide is also not critical for substrate specificity. In contrast, certain mutants in the c-region of the signal peptide result in alternate site cleavage by both Lep and SipS enzymes.

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at ؊1 (P1) and aliphatic residues at the ؊3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the ؊1 position (Karla, Proteins destined for secretion are synthesized in a precursor form with an amino-terminal extension peptide that targets the exported protein to the Sec machinery (1) or the Tat machinery (2) in bacteria. During the export process, the signal peptide is cleaved from the precursor protein by a signal peptidase that is embedded in the plasma membrane.AIn Escherichia coli, signal peptidase (SPase I) 2 consists of a single polypeptide chain of 37 kDa (3). This enzyme spans the membrane twice with a small cytoplasmic segment (residues 29 -58) and a large carboxyl-terminal catalytic domain located in the periplasm (residues 77-323) (4 -6). Catalysis by SPase I is carried out by a Ser-Lys dyad (7-10). In the case of the E. coli SPase I, Ser-90 is the nucleophilic residue that attacks the scissile bond of the precursor substrate and lysine 145 is the general base that deprotonates the serine residue (for review, see Ref. 11). A critical serine and lysine residue is also present in SPases from other species of bacteria (12), and members of the signal peptidase I family in mitochondria (13).With the exception of the mitochondrial inner membrane peptidase I (Imp1), all type I signal peptidases carry out processing with a specificity for small aliphatic residues at the Ϫ1 (P1) and Ϫ3 (P3) positions (11). Alanine is usually the preferred amino acid residue at the Ϫ1 and Ϫ3 positions and results in the frequently observed "Ala-X-Ala" motif for signal peptide cleavage (14 -16). The residues of SPase I that comprise the substrate binding site have been identified by solving the x-ray structure of the soluble catalytic domain with a covalently attached 5S penem inhibitor (10) and a structure with a non-covalent lipohexapeptide inhibitor (17). The three-dimensional structure of SPase I with no inhibitor bound (apo-structure) revealed that there is some variation in the binding pocket volume when compared with the inhibitor-bound structures (18). The E. coli SPase residues making direct van der Waals contact with the P1 methyl group are . Those making contact with the P3 residues are Phe-84, Ile-144, Val-132, and Ile-86. The substrate binding to SPase I occurs in an extended conformation. Recently, we have made mutations of the E. coli SPase I in the S1 and S3 pockets that bind the P1 and P3 residues of the substrate to identify the residues that control the substrate specificity (19). We found that alterations of the Ile-144 and Ile-86 residues to alanine residues could alter the substrate specificity and lead to cleava...