Preserved hydride transfer mechanism in evolutionarily divergent thymidylate synthases.

Thymidylate synthase (TSase) catalyzes a hydride transfer in the last step of the biosynthesis of the DNA nucleotide thymine. We compared two isozymes, namely, TSase from (TSase) and TSase from (TSase) that represent a case of divergent evolution. Interestingly, a highly conserved histidine (H147 of TSase) was proposed to serve a critical role in catalysis, but in TSase it is naturally substituted by valine (Val). Yet, TSase is more active than TSase, and the intrinsic kinetic isotope effects (KIEs) of both are temperature-independent, suggesting a similarly well-organized transition state (TS) for the catalyzed hydride transfer. To examine the role of that histidine (His) in TSase catalysis, we examined the kinetics of H147V TSase, which "bridges" between these two TSases. In contrast to both wild-type TSases, the single mutation results in deficient catalysis. The mutation leads to intrinsic KIEs that are temperature-dependent, indicating a substantial imperfection in its TS. The findings reveal two important features: a direct role of H147 in the hydride transfer step catalyzed by the TSase and the evolutionary compensation for its deficiency in TSase extensive polymorphism across the protein. Very different active site residues are observed for these evolutionarily divergent isozymes, which result in a well-organized TS for both. It is suggested that evolutionary pressure compensated for the H to V substitution at the active site of TSase by polymorphism leading to a well-organized TS in both enzymes.