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

作者：Varga Balazs; Barabas Orsolya; Takacs Eniko; Nagy Nikolett; Nagy Peter; Vertessy Beata G^*

来源：Biochemical and Biophysical Research Communications, 2008, 373(1): 8-13.

DOI：10.1016/j.bbrc.2008.05.130

• tuberculosis
• Mycobacterium tuberculosis
• dUTPase
• tryptophan sensor
• pyrophosphate assay
• inhibitor screening
• fluorescence spectroscopy
• enzyme kinetics
• uracil

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 1.5 angstrom 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.

• 出版日期2008-8-15