title: Preserved hydride transfer mechanism in evolutionarily divergent thymidylate synthases.
abstract: Thymidylate synthase (TSase) catalyzes a hydride transfer in the last step of thede novobiosynthesis of the DNA nucleotide thymine. We compared two isozymes, namely, TSase fromEscherichia coli(ecTSase) and TSase fromBacillus subtilis(bsTSase) that represent a case of divergent evolution. Interestingly, a highly conserved histidine (H147 ofecTSase) was proposed to serve a critical role in catalysis, but inbsTSase it is naturally substituted by valine (Val). Yet,bsTSase is more active thanecTSase, 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 H147VecTSase, 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 theecTSase and the evolutionary compensation for its deficiency inbsTSaseviaextensive 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 ofbsTSase by polymorphism leading to a well-organized TS in both enzymes.
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Thelma
authors.initials: T
authors.lastname: Abeysinghe
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA; Genentech, South San Francisco, CA 94080, USA.
authors.forename: Baoyu
authors.initials: B
authors.lastname: Hong
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA; Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Amnon
authors.initials: A
authors.lastname: Kohen
keywords: evolution; hydride transfer; isotope effect; marcus models; thymidylate synthase