Molecular Dynamics-Guided Engineering of Penicillin G Acylase for Enhanced β-Lactam Biosynthesis using Bacillus megaterium

Penicillins and cephalosporins are crucial in treating bacterial infections, but the processes used in producing them via chemical synthesis are environmentally unfriendly and expensive. Using enzymes such as penicillin G acylase (PGA) might be a greener option. However, natural forms of this enzyme can be inefficient in some applications involving synthesis pathways. In this case, we aimed to test the effects of a specific single point mutation, βThr68→βTyr68, on the extracellular PGA from Bacillus megaterium, focusing on its functional improvements. To understand the effects of this mutation, we conducted various computational analyses, including homology modelling, docking simulations, and the construction of three-dimensional models using molecular dynamics (MD) simulations over a 100-ns runtime. Unexpectedly, preliminary docking results suggested that the wild-type enzyme outperformed the substrate binding by more than 9.1 kcal/mol, while the mutant bound to roughly 7.6 kcal/mol. Also, when it comes to MM-PBSA, the binding free energy of the mutant gave better results, being more negative, meaning more thermodynamic stability (–28.02 ± 30.51 kJ/mol) because of stronger electrostatic interactions than its peers. Both enzyme forms were steady during the MD simulations. The mutant was equal or superior in behaviour metrics like RMSD, flexibility, surface exposure, hydrogen bonding, free energy landscapes and principal component analysis, validating the mutant's structural constancy over time. The mutant also demonstrated stronger protein–protein interactions. Collectively, these results support the fact that the mutation increases the structural and thermodynamic properties, making the mutated BmPGA a viable option for effective β-lactam antibiotic production.