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

Journal Article · Fri Aug 15 04:00:00 EDT 2008 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2008.05.130· OSTI ID:21143816

Varga, Balazs; Barabas, Orsolya; Takacs, Eniko; Nagy, Nikolett; Nagy, Peter ^[1]

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1. Laboratory 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{sup 2+} at 1.5 A 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{sub 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.

• OSTI ID: 21143816
• Journal Information: Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 373; ISSN 0006-291X; ISSN BBRCA9
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
BERYLLIUM 10
BIOSYNTHESIS
CATALYSIS
CRYSTAL STRUCTURE
DNA
ENZYMES
FLUORESCENCE SPECTROSCOPY
HISTIDINE
IMIDES
MAGNESIUM IONS
MYCOBACTERIUM TUBERCULOSIS
PYROPHOSPHATES
SCREENING
SENSORS
TRYPTOPHAN
TUBERCULOSIS
URACILS