Role of two conserved water molecules in the catalytic pocket of thymidylate synthase from Mycobacterium tuberculosis.

Thymidylate synthase from Mycobacterium tuberculosis (MtbThyX) is a key enzyme and a promising target for drug development. While specific inhibitors are not yet available, research in this area is crucial. We used alchemical free energy simulations to quantify the stabilising effect of two conserved buried water molecules in the polar active site of MtbThyX, ensuring its structural integrity. Removing one of these localised water molecules quickly opens a lid, allowing another water molecule to enter and seal the active site. The conserved water sites are accessed through two distinct channels, with an arginine residue or the cofactor FAD serving as lids. The properties of these conserved water molecules differ significantly from bulk water, and their removal raises the free energy of the MtbThyX complex by as much as 12 kcal mol-1, triggering rapid lid opening without additional energy input. This reversible mechanism resembles prey capture in pitcher plants. With about 8 µs of sampling from multiple runs, we achieved good convergence and a statistical uncertainty of around 0.5 kcal mol-1 for the estimated free energies.