Maturation of Pseudomonas aeruginosa Elastase

Abstract

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. After propeptide-mediated folding in the periplasm, the proenzyme is autoproteolytically processed, prior to translocation of both the mature enzyme and the propeptide across the outer membrane. The formation of the two disulfide bonds present in the mature enzyme was examined by studying the expression of the wild-type enzyme and of alanine for cysteine mutant derivatives in the authentic host and in dsb mutants of Escherichia coli. It appeared that the two disulfide bonds are formed successively. First, DsbA catalyzes the formation of the disulfide bond between Cys-270 and Cys-297 within the proenzyme. This step is essential for the subsequent autoproteolytic processing to occur. The second disulfide bond between Cys-30 and Cys-57 is formed more slowly and appears to be formed after processing of the proenzyme, and its formation is catalyzed by DsbA as well. This second disulfide bond appeared to be required for the full proteolytic activity of the enzyme and contributes to its stability.The opportunistic pathogen Pseudomonas aeruginosa secretes many proteins into the extracellular medium. The secreted proteins are synthesized in the cytoplasm and have to pass both membranes of the cell envelope. Four main pathways for the secretion of proteins, usually referred to as the type I, II, III, and the autotransporter pathway, have been identified in P. aeruginosa (1-4). The majority of the exoproteins characterized is secreted via the type II pathway, also referred to as the general secretory pathway. In addition to elastase, which is the most abundant secreted protein, lipase, alkaline phosphatase, exotoxin A, two phospholipases C, the staphylolytic protease LasA, the chitin-binding protein CbpD, and a putative aminopeptidase are secreted via this pathway (2, 5-7).Proteins secreted via the type I or type III pathways are translocated across the two membranes of the cell envelope in a single step, without a periplasmic intermediate. In contrast, the type II pathway is a two-step mechanism. The first step is the translocation across the inner membrane, which is mediated by the Sec machinery. In the periplasm, which contains chaperones and folding catalysts, the exoproteins fold into a (near-)native conformation (8). For several proteins that are secreted via a type II mechanism (9 -11), including elastase of P. aeruginosa (12,13), it has been demonstrated that folding in the periplasm is essential for the subsequent translocation across the outer membrane to occur. In P. aeruginosa, the translocation of the periplasmic intermediates across the outer membrane is mediated by a machinery composed of at least 12 proteins, encoded by the xcp genes (for a review, see Ref. 2).To investigate the biogenesis of proteins secreted via the type II pathway of P. aeruginosa, we have chosen elastase as a model. This metalloprotease, which is encoded by the lasB gene (14), is produced as a preproprotein. The pre-part is the signal peptide, which directs the translo...

A Secreted Aminopeptidase of Pseudomonas aeruginosa

“…The identification of a putative signal peptide in PaAP suggests that it is secreted via the general secretion pathway, which requires the Xcp machinery (50). In support of this, the band corresponding to the putative aminopeptidase (AP 58 ) is not detectable in the culture medium of an xcp mutant of P. aeruginosa (49).…”

“…A hint that P. aeruginosa may secrete an aminopeptidase came from a recent study by Braun et al (49) in which the authors have shown that the N-terminal sequence of a 58-kDa protein secreted by a P. aeruginosa mutant lacking both elastase and Apr corresponds to an unknown protein in the P. aeruginosa data base that exhibits 28 Aminopeptidase activity towards various amino acid p-nitroanilide derivatives was measured with 0.4 mM substrate and 2 g of AP 28 as described under "Experimental Procedures." pNA, para-nitroanilide.…”

“…In this study, we describe for the first time an aminopeptidase secreted by P. aeruginosa. Examination of the primary structure of PaAP reveals that the sequence APSEAQQFTE found previously (49) for the N terminus of the 58-kDa putative aminopeptidase (designated below AP 58 ) corresponds to positions 25-34 in the deduced amino acid sequence of PaAP (Fig. 4A).…”

The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC

“…This "post-translocational" disulfide bond formation is dependent on the folding of elastase. Tommassen and co-workers (39) have demonstrated that the N-terminal disulfide bonds of elastase are not formed until the proper folding of the C terminus is achieved resulting in autoprocessing of the elastase proenzyme. Thus, some disulfide bonds may only be formed once the protein achieves a certain tertiary structure, which does not occur until the C-terminal disulfide bond-dependent autoprocessing has occurred.…”

“…First, in the case of the metalloprotease elastase from Pseudomonas aeruginosa, DsbA catalyzes the formation of a C-terminal disulfide bond (Cys 270 -Cys 297 ) prior to the formation of the N-terminal disulfide bond (Cys 30 -Cys 57 ) (39). This "post-translocational" disulfide bond formation is dependent on the folding of elastase.…”