Functional and structural analyses of amino acid sequence variation in PDC β-lactamase reveal different mechanistic pathways toward cefiderocol resistance in <i>Pseudomonas aeruginosa</i>

A wide variety of clinically observed amino acid alterations in the <i>Pseudomonas aeruginosa</i> chromosomal β-lactamase AmpC (<i>Pseudomonas</i>-derived cephalosporinase [PDC]) are associated with increased resistance to cefepime, ceftolozane/tazobactam, or ceftazidime/avibactam, but their impact on cefiderocol resistance is unclear. We took advantage of a previously engineered collection of wild-type (PAO1) and iron uptake-deficient (PAO Δ<i>piuC</i>) <i>P. aeruginosa</i> isolates producing 19 distinct PDC variants with substitutions in key catalytic regions. While most variants had moderate effects on cefiderocol minimum inhibitory concentrations compared to PDC-1, the E219K (Ω-loop) and L293P (helix H10) variants significantly affected cefiderocol activity. Kinetic studies revealed that both mutations improve cefiderocol hydrolysis through different enzymatic mechanisms compared to PDC-1 (<i>K</i>_m = 85.29 µM, <i>k</i>_cat = 0.0036 s^-1, and <i>k</i>_cat/<i>K</i>_m = 0.00004 µM^-1 s^-1), leading to enhanced turnover in PDC E219K (<i>K</i>_m = 465.64 µM, <i>k</i>_cat = 0.45 s^-1, and <i>k</i>_cat/<i>K</i>_m = 0.00096 µM^-1 s^-1) and improved affinity in PDC L293P (<i>K</i>_m = 2.69 µM, <i>k</i>_cat = 0.0036 s^-1, and <i>k</i>_cat/<i>K</i>_m = 0.00135 µM^-1 s^-1). These mechanisms are also involved in resistance to ceftolozane and cefepime, identified as the preferred substrates for the E219K and L293P variants, respectively. Molecular dynamics (MD) simulation studies revealed that (i) rigidification of the Ω-loop in PDC E219K promotes optimal accommodation of the R¹ group of cefiderocol, enhancing nucleophilic attack by the catalytic serine; (ii) the less folded conformation of helix H10 in PDC L293P improves cefiderocol accommodation in the active site by establishing stronger hydrogen-bonding interactions with the R² group. Our findings demonstrate that the PDC β-lactamase may take advantage of the structural similarities between cefiderocol and other cephalosporins and accelerate hydrolysis by accommodating the E219K or L293P amino acid replacements.