Structural and functional characterization of PDC-16 β-lactamase reveals a constrained active-site architecture associated with altered catalysis

The emergence of β-lactam resistance in Pseudomonas aeruginosa is driven in part by diversification of Pseudomonas-derived cephalosporinases (PDCs), particularly extended-spectrum AmpC variants that typically acquire broader substrate profiles through active-site expansion. Here, we report the structural and functional characterization of the PDC-16 variant of Pseudomonas aeruginosa β-lactamase through high-resolution crystal structures of its apo form (2.30 Å) and imipenem-bound complex (2.20 Å), complemented by kinetic and computational analyses. Contrary to the established paradigms of active-site expansion, PDC-16 exhibits a reduced active-site cavity volume (351.22 Å3) and decreased polarity, compared to the wild-type PDC-1 (371.74 Å3) and cavity expanded PDC-3 (405.43 Å3), arising from subtle repositioning of cavity-lining residues (Met266, Asn288, and Thr320) and Ω-loop (~S152-Y200)-mediated conformational constraints. The imipenem-bound structure captures a covalent acyl-enzyme intermediate with Ser64 and shows a conformational rearrangement of the Y200-R212 region, which adopts β3-β4 strand-like geometry in the ligand-bound crystallographic state. This observation is consistent with ligand-associated remodeling of the active-site architecture, although the crystal structures alone do not establish the dynamic mechanism underlying this transition. Consistent with these structural features, kinetic analyses revealed reduced catalytic efficiency toward ampicillin and carbapenems, while activity toward cefotaxime was comparatively preserved, indicating substrate-dependent effects associated with the constrained active-site architecture. Biochemical assays showed that avibactam produced substantially greater inhibition of PDC-16 than representative phenazine-derived compounds, underscoring that favorable computational binding does not necessarily translate into inhibitory efficacy. Together, these findings support an association between the remodeled active-site architecture and substrate-dependent catalytic behavior in PDC-16.