Dissecting the roles of aminopeptidases and aminoacyl tRNA-protein transferases in the N-End rule of Pseudomonas aeruginosa

Bacteria live in dynamic environments. Tight control of proteolysis ensures adaptability, allowing cells to control levels of short-lived or damaged proteins, remodel multisubunit complexes, and propagate signals. Proteins are targeted for degradation by the addition of destabilising signals termed degrons. Degrons may be located internally or terminally, comprising individual residues, peptides, or posttranslational modifications. The N-end rule describes single-residue degrons at the N-terminus, correlating the in vivo half-life of a protein with its N-terminal residue. This rule dictates that bacterial proteins possessing N-terminal leucine or phenylalanine undergo rapid proteolysis. This work focuses on the characterisation of three enzymes from Pseudomonas aeruginosa which add or remove leucine or phenylalanine from N-termini. Leucyl/phenylalanyl-tRNA protein transferase (LFT) and bacterial protein transferase (BPT) conjugate leucine and phenylalanine to substrate N-termini, while leucine/phenylalanine aminopeptidases (LF-APs, including PaPepA) cleave N-terminal leucine and phenylalanine residues. It is not understood whether LF-APs work alongside or competitively with LFT and BPT, or how their peptidase activity is regulated. Furthermore, it is unknown whether the production and activities of LFT, BPT, and PaPepA are regulated by metabolic state, and the mechanisms underpinning their substrate selection are not understood. This work sought to develop methods to identify putative LFT and BPT substrates, to shed light on their substrate selectivity and reveal how these enzymes feed into the wider process of protein degradation. Initial investigation revealed that BPT and LFT can transfer unnatural amino acids (UAAs) which are amenable to click chemistry. This finding was used to detectably tag prospective N-end rule substrates by performing click chemistry experiments on P. aeruginosa lysates. This work also investigated catalysis by a leucyl aminopeptidase, PaPepA, probing its metal ion binding and activation, substrate selection, and catalytic mechanism. The findings presented will facilitate a deeper understanding of metalloenzymes and processive peptidases and proteases in protein homeostasis.