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

• Published in: Biochemical and biophysical research communications 2008-08, Vol.373 (1), p.8-13
• Main Authors: Varga, Balázs, Barabás, Orsolya, Takács, Enikő, Nagy, Nikolett, Nagy, Péter, Vértessy, Beáta G.
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; Amino Acid Sequence; Antitubercular Agents - chemistry; Antitubercular Agents - isolation & purification; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; BERYLLIUM 10; Binding Sites - genetics; BIOSYNTHESIS; CATALYSIS; CRYSTAL STRUCTURE; Crystallography, X-Ray; Deoxyuracil Nucleotides - chemistry; DNA; dUTPase; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - isolation & purification; Enzyme kinetics; ENZYMES; Fluorescence; FLUORESCENCE SPECTROSCOPY; HISTIDINE; IMIDES; Inhibitor screening; Magnesium - chemistry; MAGNESIUM IONS; Microbial Sensitivity Tests; Molecular Sequence Data; MYCOBACTERIUM TUBERCULOSIS; Mycobacterium tuberculosis - enzymology; Protein Conformation; Pyrophosphatases - chemistry; Pyrophosphatases - genetics; Pyrophosphate assay; PYROPHOSPHATES; SCREENING; SENSORS; TRYPTOPHAN; Tryptophan - analysis; Tryptophan - chemistry; Tryptophan - genetics; Tryptophan sensor; TUBERCULOSIS; Uracil; URACILS
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2008.05.130

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 Mg 2+ 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. K d 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.