Abstract
The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. Using reinforcement learning-driven molecular dynamics simulations and constant pH MD, we investigate how clinically observed mutations in the Ω-loop (at residues V211, G214, E219, and Y221) alter the structure and function of PDC-3. Our findings reveal that these substitutions modulate the dynamic flexibility of the Ω-loop and the R2-loop, reshaping the cavity of the active site. In particular, E219K and Y221A disrupt the tridentate hydrogen bond network around K67, thus lowering its pKa and promoting proton transfer to the catalytic residue S64. Markov state models reveal that E219K achieves enhanced catalysis by adopting stable, long-lived 'active' conformations, whereas Y221A facilitates activity by rapidly toggling between bond-formed and bond-broken states. In addition, substitutions influence key hydrogen bonds that control the opening and closure of the active-site pocket, consequently influencing the overall size. The pocket expands in all nine clinically identified variants, creating additional space to accommodate bulkier R1 and R2 cephalosporin side chains. Taken together, these results provide a mechanistic basis for how single residue substitutions in the Ω-loop affect catalytic activity. Insights into the structural dynamics of the catalytic site advance our understanding of emerging β-lactamase variants and can inform the rational design of novel inhibitors to combat drug-resistant P. aeruginosa.