3 years ago

Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate

Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate
Kanagaraj Sekar, Ansuman Biswas, Arpit Shukla, Jeyaraman Jeyakanthan, Rajendran Santhosh, Santosh Kumar Chaudhary
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. Thymidylate kinase (TMK) is an important enzyme for DNA synthesis. The study reports the different conformational substates along the reaction pathway of a hyperthermophilic TMK from Aquifex aeolicus. The analyses highlight the role of an Arg residue in the Lid region in catalysis, and provide insights into the order of substrate binding and phosphate transfer reaction mechanism.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1111/febs.14140

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