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

2008; Elsevier BV; Volume: 373; Issue: 1 Linguagem: Inglês

10.1016/j.bbrc.2008.05.130

1090-2104

Balázs Varga, O. Barábas, Enikő Takács, Nikolett Nagy, Péter Nagy, Beáta G. Vértessy,

Enzyme Structure and Function

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, α,β-imido-dUTP and Mg2+ at 1.5 Å 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. Kd for α,β-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.