Thymidylate kinase (TMK) is a key enzyme which plays an important role in DNA synthesis. It belongs to the family of nucleoside monophosphate kinases, several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three-dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal-mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild-type protein and mutants. Furthermore, the enzyme was found to exhibit broad specificity toward phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes.
Database: Structural data are available in the PDB under the accession numbers 2PBR, 4S2E, 5H5B, 5XAI, 4S35, 5XB2, 5H56, 5XB3, 5H5K, 5XB5, and 5XBH.
Keywords: active site dynamics; associative phosphoryl transfer; random bi-bi mechanism; thymidylate kinase; transition state.
© 2017 Federation of European Biochemical Societies.