Structural and functional characterization of diadenosine tetraphosphate hydrolase (ApaH) from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen responsible for life-threatening infections, particularly in cystic fibrosis patients, that poses a major global health challenge due to its intrinsic and acquired antibiotic resistance. The urgent need for alternative therapeutic approaches has shifted attention toward antivirulence strategies aimed at disarming pathogenicity rather than inhibiting bacterial growth. Among emerging regulatory pathways, the alarmone-like molecule diadenosine tetraphosphate (Ap4A) has recently been identified as a key regulator of P. aeruginosa virulence. The enzyme diadenosine tetraphosphate hydrolase (ApaH) is responsible for Ap4A degradation; however, no structural or functional information was previously available for its P. aeruginosa counterpart (PaApaH). This thesis reports the first biochemical and structural characterization of PaApaH. The enzyme was successfully purified in a monodisperse and stable form and extensively characterized. Enzymatic assays demonstrated that PaApaH is a metal-dependent symmetrical Ap4A hydrolase with a moderate catalytic turnover. The enzyme displays a clear preference for Mn²⁺, showing optimal activity under near-physiological conditions and selectivity for Ap4A over other dinucleotide polyphosphates. Three high-resolution crystal structures of PaApaH were solved in complex with distinct metal cofactors (Mn²⁺ and Mg²⁺) and with its hydrolysis product ADP. Structural analyses revealed a conserved bimetallic active site and allowed the identification of the molecular basis of metal dependence. Structural comparison and in silico analyses enabled the identification of residues involved in substrate binding and catalysis. Site-directed mutagenesis of these residues, followed by in vitro enzymatic assays and in vivo phenotypic analyses in P. aeruginosa, revealed new functional determinants whose role in catalysis or substrate binding had never been described before. Characterization of these variants established a clear link between ApaH enzymatic activity and the virulent phenotype of P. aeruginosa.Overall, this study provides the first biochemical and structural characterization of ApaH from P. aeruginosa. The results validate PaApaH as a promising molecular target for the rational development of selective antivirulence therapeutics, paving the way for new strategies to counteract multidrug-resistant P. aeruginosa infections.