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J Biol Chem. 2016 Dec 16;291(51):26320-26331. Epub 2016 Nov 4.

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes.

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From the Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary H1117.
the Institute of Chemistry, University of Miskolc, Miskolc, Hungary H3529.
the Department of Applied Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary H1111, and.
Department of Chemical Informatics, University of Szeged, Szeged, Hungary H6725.
From the Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary H1117,


dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent.


Mycobacterium tuberculosis; X-ray crystallography; dUTPase; molecular dynamics; molecular modeling; nucleotide; pre-steady-state kinetics; solvent accessibility; substrate binding

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