Decoding the full chemical catalytic cycle of class I PHA synthase: A QM/MM metadynamics perspective

The accumulation of plastic waste continues to pose a significant threat to ecosystems, driven by inefficient recycling processes and inadequate waste management. Polyhydroxyalkanoates (PHAs) have emerged as promising biodegradable alternatives, owing to their ability to degrade under various environmental conditions and modulable material properties, which enable broad applicability. Despite these advantages, the detailed molecular mechanisms of PHA biosynthesis-particularly those of the key enzyme PHA synthase (PhaC)-have remained unclear, often limiting efforts to improve its functionality. In this study, we investigated the catalytic mechanism of Class I PHA synthase from Cupriavidus necator using Quantum Mechanics/Molecular Mechanics (QM/MM) metadynamics at the PBE/MM Molecular Dynamics (MD) level. A large QM region was used to capture key interactions during the polymerization of 3-hydroxybutyryl-CoA (3HB-CoA) monomers. Our results identified the initial nucleophilic attack by Cys319, occurring concurrently with proton transfer to His508. This first nucleophilic attack was identified as the rate-limiting step, with an activation barrier of 11.6 kcal·mol^-1. Comparative analysis of elongation pathways mediated by His508 and Asp480 showed that the Asp480-mediated pathway is energetically more favorable. These findings provide mechanistic insights into PHA polymerization and establish a foundation for the future development of more efficient PHA synthases.