Proteolytic Cleavage of a C-terminal Prosequence, Leading to Autoprocessing at the N Terminus, Activates Leucine Aminopeptidase from Pseudomonas aeruginosa

Abstract

At high bacterial cell density the gene expression program of Pseudomonas aeruginosa is regulated by quorum sensing. Among the gene products highly up-regulated by this system is an exoprotease, leucine aminopeptidase (PA-LAP), which is coexpressed with several known virulence factors and secreted as a proenzyme. We undertook a study of its activation by expressing the full-length proform of PA-LAP recombinantly in Escherichia coli (here termed, rLAP55) and characterizing individual steps in its conversion to an active enzyme. Activation is initiated with the proteolytic removal of a C-terminal prosequence. Removal of ϳ20 amino acids is accomplished by Pseudomonas elastase, which is also positively regulated by quorum sensing. Activation is also mediated by other proteases that cleave rLAP55 near its C terminus. The importance of the C terminus was confirmed by showing that C-terminal deletions of 1-24 amino acids produce a fully active enzyme. The removal of C-terminal prosequences either by proteolysis or deletion leads to an unusual autoprocessing event at the N terminus. Autoprocessing is apparently an intramolecular event, requires the active site of LAP, and results in the removal of 12 N-terminal amino acids. Furthermore, a detailed analysis of the C-terminal prosequence suggests that the proenzyme state is dependent on the presence of a basic side chain contributed by the last amino acid, lysine 536. Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus.

Extracellular aminopeptidase modulates biofilm development of Pseudomonas aeruginosa by affecting matrix exopolysaccharide and bacterial cell death

“…aeruginosa aminopeptidase (PaAP, we designated PA2939 as paaP in this study) (Cahan et al ., ). PaAP is secreted as a precursor and is later processed into an active enzyme in culture supernatants (Sarnovsky et al ., ). Its active site likely includes the amino acid residues His‐296, Asp‐308, Glu‐341, Asp‐369 and His‐467 (Cahan et al ., ).…”

Vesicle-associatedPseudomonas aeruginosaleucine aminopeptidase modulates bacterial biofilms grown on host cellular substrates

“…To further corroborate our finding that soluble forms of PaAP cannot prevent microcolony development, we purified and refolded recombinant P. aeruginosa aminopeptidase (rPaAP) expressed in E. coli 14 . The specific activity of this preparation was similar to, though slightly lower than, that of the native soluble enzyme purified from culture supernatants.…”

“…rPaAP expression, purification, and refolding were carried out as described previously 14 , and the refolded protein was concentrated using 10,000 MWCO Amicon filter units (Millipore-Sigma).…”

“…Initially, PaAP enters the extracellular environment as a 536 amino acid proenzyme. It is then processed by a variety of extracellular proteases at the C terminus to activate its enzymatic activity, and finally undergoes auto-processing at its N-terminus to create a 56 kDa, enzymatically active product 14 .…”

Hydrolysis of Amides

“…Recently, the processing of this aminopeptidase, which is secreted as a proenzyme, has been elucidated. The two-step maturation process starts with the proteolytic cleavage (e.g., by elastase) of the aminopeptidase proenzyme at its C terminus, followed by an intramolecular autocatalytic removal of the 12-amino acid propeptide from its N-terminus [195]. Many leucine aminopeptidases from parasites have been isolated and characterized in the last decade, because these enzymes are seen as a potential target for new drugs.…”