A Computational Tool for Taxadiene Synthase Mutation Site Screening

Taxol is a high value pharmaceutical who’s potent antitumoral properties have been used to treat over one million patients since its discovery. Efforts have been made to optimize the Taxol biosynthesis pathway in engineered yeast strains by identifying a mutated variant of the bottleneck enzyme, taxadiene synthase (TXS), that exhibits improved product specificity and catalytic activity. This project focused on creating a model of TXS activity that can be used as a computational tool for screening mutation site candidates. The 3D structure of the active enzyme was constructed using homology modeling. It includes a previously unresolved N-terminal region and “JK-loop” which serve the purpose of ensuring that the substrate remains in the active site during catalysis. Following this, quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations were performed for the active TXS model docked with the initial substrate, the five intermediates, and the two taxadiene isomer products. The resulting models reveal that different sets of residues interact with each of the different substrates. Furthermore, two mutations sites that were previously shown to inactivate TXS were found to play a key role in stabilizing one of the intermediates. These findings demonstrate how these models can be used as a tool for screening mutation site candidates before wet-lab testing, greatly accelerating the discovery pipeline.