Molecular dynamics-guided engineering of penicillin G acylase for enhanced β-lactam biosynthesis

Penicillins and cephalosporins are crucial in treating bacterial infections, but conventional chemical synthesis routes for their production are costly and environmentally burdensome. Enzymatic alternatives, such as penicillin G acylase (PGA), offer a greener path; however, naturally occurring forms of this enzyme often exhibit suboptimal thermodynamic stability and electrostatic complementarity under industrial synthesis conditions. In this study, we investigated the effects of a single point mutation, βThr68→βTyr68, in the extracellular PGA from Bacillus megaterium (BmPGA), with the primary objective of improving binding free energy and structural stability rather than maximising raw docking affinity. To characterise the mutation’s impact, we performed homology modelling, molecular docking, and 100-ns molecular dynamics (MD) simulations coupled with MM-PBSA binding free energy calculations. It is important to note that while molecular docking provides a rapid, pose-based approximation of binding geometry, it does not account for solvent effects, conformational entropy, or electrostatic solvation factors that are critical in enzyme substrate systems. Accordingly, MM-PBSA was prioritised as the more physically rigorous metric for evaluating binding thermodynamics. Docking scores indicated that the wild-type enzyme exhibited stronger pose-based affinity (–9.1 kcal/mol) compared to the mutant (–7.6 kcal/mol); however, this difference reflects altered binding geometry rather than reduced thermodynamic favourability. MM-PBSA analysis revealed that the mutant achieved a more negative binding free energy (–28.02 ± 30.51 kJ/mol), driven predominantly by enhanced electrostatic interactions, indicating superior thermodynamic stability of the enzyme–substrate complex. Both enzyme variants remained structurally stable throughout MD simulations, with the mutant performing comparably or better across RMSD, flexibility, solvent-accessible surface area, hydrogen bonding, free energy landscape, and principal component analyses. The mutant also exhibited stronger protein–protein interaction profiles. Taken together, these findings demonstrate that the βThr68→βTry68 substitution meaningfully enhances the thermodynamic and structural properties of BmPGA, establishing it as a promising candidate for more efficient and robust enzymatic production of β-lactam antibiotics.