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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.

Abstract

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.

PMID:
18519027
[PubMed - indexed for MEDLINE]
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