Metal binding is not enough: characterisation of aminopeptidase PepA from Pseudomonas aeruginosa Martha Simpson and Clarissa Melo Czekster The University of St Andrews, UK Protein homeostasis encompasses protein synthesis, translocation, folding, and degradation. Tight regulation of this process is essential for the survival and function of all cellular organisms. Leucine aminopeptidases (LAPs) are metallopeptidases that cleave N-terminal leucine residues from substrate proteins and peptides. These enzymes are implicated in a variety of key processes including protein degradation and maturation, metabolism of biologically active peptides, and antigen presentation 1 . LAPs are members of either the M1 or M17 protease families. M17 LAPs are highly conserved with well characterised structures. Microbial M17 LAPs have also been demonstrated to possess DNA binding functions in regulatory roles. In the case of Pseudomonas aeruginosa, PepA works as a transcriptional repressor of AlgD: the first gene in the alginate biosynthetic operon. This has important pathological ramifications as P. aeruginosa isolates from the lungs of cystic fibrosis patients exhibit dramatic increases in alginate expression 2 . Although PepA from P. aeruginosa is implicated in these key processes, it is poorly understood. Whether its main role lies primarily in protein turnover or in transcriptional regulation is not known, and its mechanism, substrate selectivity, and metal dependency are uncharacterised. Our work therefore investigates PepA from PA14 (PaPepA). We determined structures for PaPepA in its apo form, bound to metals and a substrate mimic, demonstrating it is hexamer that binds two metal ions and a substrate molecule per monomer. We have determined which metals PaPepA uses to perform its catalytic function, and probed the stoichiometry of metal binding using ITC, EPR, and stopped flow spectrofluorimetry. Our work demonstrated PaPepA function is regulated by metal activation. Metal binding in PaPepA is tight and favours manganese, in contrast to what is observed in other homologues. Prior work on substrate specificity has focussed on model substrates such as Leu- p-nitroanilide (Leu-pNA). We have shown that the kinetics of substrate turnover strongly depend on the substrate and leaving group. Different substrate preferences and kinetic properties were observed when comparing cleavage of amino acid-pNA conjugates, amino acid- 7-Amino-4-methylcoumarin (AMC) conjugates, and peptides. More broadly, our project will elucidate how the intracellular peptidase PepA feeds into the wider processes of protein turnover and homeostasis in P. aeruginosa. This can lead to novel therapeutics and wider understanding of P. aeruginosa biology, as well as crucial insights into metal binding sites and their specificity and design . Reference 1. Kale, A., Pijning, T., Sonke, T., Dijkstra, B. W., & Thunnissen, A. M. W. H. (2010). Crystal structure of the leucine aminopeptidase from pseudomonas putida reveals the molecular basis for its enantioselectivity and broad substrate specificity. Journal of Molecular Biology , 398 (5). 2. Woolwine, S. C., & Wozniak, D. J. (1999). Identification of an Escherichia coli pepA homolog and its involvement in suppression of the algB phenotype in mucoid Pseudomonas aeruginosa. Journal of Bacteriology , 181 (1).
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