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

Biswas, Ansuman and Shukla, Arpit and Chaudhary, Santosh Kumar and Santhosh, Rajendran and Jeyakanthan, Jeyaraman and Sekar, Kanagaraj (2017) Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate. In: FEBS JOURNAL, 284 (15). pp. 2527-2544.

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Abstract

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 normalmode 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.

Item Type:	Journal Article
Publication:	FEBS JOURNAL
Additional Information:	Copy right for this article belongs to the WILEY, 111 RIVER ST, HOBOKEN 07030-5774, NJ USA
Department/Centre:	Division of Interdisciplinary Sciences > Supercomputer Education & Research Centre Division of Physical & Mathematical Sciences > Physics
Date Deposited:	01 Sep 2017 09:12
Last Modified:	01 Sep 2017 09:12
URI:	http://eprints.iisc.ac.in/id/eprint/57712

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