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Biochem Biophys Res Commun. 2008 Aug 15;373(1):8-13. doi: 10.1016/j.bbrc.2008.05.130. Epub 2008 Jun 2.

Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor.

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  • 1Laboratory of Genome Metabolism and Repair, Institute of Enzymology, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest, Hungary.


dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, alpha,beta-imido-dUTP and Mg(2+) at 1.5A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K(d) for alpha,beta-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.

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