, Volume 72, Issue 3, pp 306-314

Evolution of Metamorphism in Thymidylate Synthases Within the Primate Lineages

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Abstract

Crystal structures of human thymidylate synthase (hTS) revealed that the protein exists in active and inactive conformations, defined by the position of a loop containing the active site nucleophile. TS is highly homologous among diverse species; however, the residue at position 163 (hTS) differs among species. Arginine at this position is predicted by structural modeling to enable conformational switching. Arginine or lysine is reported at this position in all mammals in the GenBank and Ensembl databases, with arginine reported in only primates. Sequence analysis of the TS gene of representative primates revealed that arginine occurs at this relative position in all primates except a representative of prosimians. Mutant human proteins were created with residues at position 163 that occur in TSs from prokaryotes and eukaryotes. Catalytic constants (k cat) of mutant enzymes were 45–149% of hTS, with the lysine mutant (R163K) exhibiting the highest k cat. The effect of lysine substitution on solution structure and on ligand binding was investigated. R163K exhibited higher intrinsic fluorescence, a more negative molar ellipticity, and higher dissociation constants (K d) for ligands that modulate protein conformation than hTS. Temperature effects on intrinsic fluorescence and catalytic activity of hTS and R163K are consistent with proteins populating different conformational states. The data indicate that the enzyme with arginine at the position corresponding to 163 (hTS) evolved after the divergence of prosimians and simians and that substitution of lysine by arginine confers unique structural and functional properties to the enzyme expressed in simian primates.

BeiBei Luo and Saphronia R. Johnson contributed equally to this research.
Primate TS sequences have been submitted to the GenBank data repository. Accession numbers are FJ821290 (Pan troglodytes), FJ821291 (Pan paniscus), FJ821292 (Gorilla gorilla), FJ821293 (Lemur catta), FJ821294 (Macaca mulatta), FJ821295 (Saimiri sciureus).