LAUSR

Thymidylate synthase is an enzyme that catalyzes deoxythymidine monophosphate (dTMP) synthesis from substrate deoxyuridine monophosphate (dUMP). Thymidylate synthase of Mycobacterium tuberculosis (MtbThyX) is structurally distinct from its human analogue human thymidylate synthase (hThyA), thus drawing attention as an attractive drug target for combating tuberculosis. Fluorodeoxyuridylate (F-dUMP) is a successful inhibitor of both MtbThyX and hThyA, thus limited by poor selectivity. Understanding the dynamics and energetics associated with substrate/inhibitor binding to thymidylate synthase in atomic details remains a fundamental unsolved problem, which is necessary for a new selective inhibitor design. Structural studies of MtbThyX and hThyA bound substrate/inhibitor complexes not only revealed the extensive specific interaction network between protein and ligands but also opened up the possibility of directly computing the energetics of the substrate versus inhibitor recognition. Using experimentally determined structures as a template, we report extensive computer simulations (∼4.5 μs) that allow us to quantitatively estimate ligand selectivity (dUMP vs F-dUMP) by MtbThyX and hThyA. We show that MtbThyX prefers deprotonated dUMP (enolate form) as the substrate, whereas hThyA binds to the keto form of dUMP. Computed energetics clearly show that MtbThyX is less selective between dUMP and F-dUMP, favoring the latter, relative to hThyA. The simulations reveal the role of tyrosine at position 135 (Y135) of hThyA in amplifying the selectivity. The protonation state of the pyrimidine base of the ligand (i.e., keto or enolate) seems to have no role in MtbThyX ligand selectivity. A molecular gate (consists of Y108, K165, H203, and a water molecule) restricts water accessibility and offers a desolvated dry ligand-binding pocket for MtbThyX. The ligand-binding pocket of hThyA is relatively wet and exposed to bulk water.