(169z) Exploration of PHA Synthase Mechanism through QM/MM Simulations

Plastic waste accumulation is exacerbating global ecosystem destruction and pollution. Despite the continuous increase in plastic production, a significant portion of plastic remains unrecycled and ends up being incinerated, landfilled, or improperly managed. Consequently, there is a growing demand for biodegradable plastics to address the regulation of non-biodegradable single-use plastic products and the escalating plastic waste crisis. Among these biodegradable plastics, Polyhydroxy alkanoate (PHA) stands out as a promising solution. Despite its advantages of adjustable decomposition periods and cost-effective raw materials, PHA production suffers from inadequate productivity. However, PHA is considered the most ideal biodegradable plastic due to its unique ability to decompose not only in soil but also in seawater, making it the only biodegradable material capable of such widespread decomposition. Therefore, synthesizing PHA is crucial, requiring optimal pretreatment and bacterial enzyme optimization, especially to avoid bacterial carbon source excess, hindering synthesis. The final enzyme used in PHA synthesis relies on a specific substance, 3-hydroxybutyryl-CoA (3HB-CoA). Currently, the catalytic site of PHA synthase is known as HIS-CYS-ASP, but the mechanism remains poorly understood. In this study, we provide an atomistic and thermodynamic interpretation of the catalytic reaction mechanism of PHA synthase using umbrella sampling simulations at the robust B3LYP/MM MD level with a large QM region. The reaction mechanism involves proton transfer from Cys118 to His307, concerted with a nucleophilic attack by Cys118 on the substrate, leading to the release of CoA. Subsequently, another 3HB-CoA is inserted into the active site, and the elongation of 3HB occurs through the same process. Additionally, we utilized Density Functional Theory (DFT) to calculate the mechanism solely for the active site and compared the results with QM/MM simulation outcomes. This work enhances our understanding of the catalytic mechanism of PHA synthase and paves the way for further rational enzyme engineering efforts.