Ntermanakis_2026_Chembiochem_27_e70424

Industrial biocatalysts are strongly influenced by their physicochemical environment, yet systematic studies on how metals and macromolecular crowding influence metal-independent esterases remain scarce. Here, Pseudomonas fluorescens esterase I (PFE), an alpha/beta-hydrolase, is employed to evaluate how divalent cations and molecular crowding modulate catalysis and structural integrity. PFE retained >85% activity for most metals at 2 mM, demonstrating notable robustness. By contrast, Cu(2+) and Zn(2+) induced pronounced inhibition, with loss of activity at higher metal concentrations. Combined activity, EDTA recovery, dynamic light scattering, and native-PAGE experiments reveal Zn(2+) and Cu(2+) perturb PFE's structure, promoting structural heterogeneity and the formation of aggregates, whereas for Cu(2+) computational predictions further support interactions with active site residues. Mg(2+), Ca(2+), Ba(2+), Mn(2+), Co(2+), and Ni(2+) exert weaker and variable effects. Unlike chemical perturbation, macro- and micromolecular crowding was idle to PFE's activity, despite increases in viscocity, underscoring resilience to physical crowding. Overall, PFE responses to metals are governed by metal-specific coordination chemistry and distinct deactivation mechanisms, providing a framework for understanding esterase performance in metal-rich and crowded environments and highlighting physicochemical tuning as a complementary strategy to protein engineering in biocatalysis.