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| Structural biology of dUTPase enzyme family: understanding the tools for uracil-DNA metabolism. | |
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dUTPase prevents uracil incorporation into DNA. Lack of the enzyme initiates
thymine-less cell death by transformation of base excision repair into a
hyperactive futile cycle. A detailed understanding of mechanism and the
cellular role of the enzyme is expected to offer innovative new means to
modulate the process of thymine-less apoptosis.
Here, structural/mechanistic aspects of enzyme action are addressed by
fruitful combination of high-resolution studies in the crystal (X-ray
crystallography), as well as in solution (multidimensional NMR,
electron-spin resonance, and electron-nuclear double resonance (EPR/ENDOR)
spectroscopies), together with kinetic and thermodynamic investigations.
Results include i) structural snapshots along the reaction coordinate of
enzymatic phosphate ester hydrolysis to identify a mechanism of significant
associative character, ii) experimenal observation of the high-energy
pentacovalent phosphorous intermediate as a direct evidence for the
central dogma of enzymology, iii) nucleotide-binding-induced alteration
of dynamics of essential protein side chains by multidimensional NMR (1),
iv) evolution of an allosteric mechanism in dUTPase (1,2), and v) practical
applications of a unique metal-ion substitution by vanadyl (VO2+) cation
for EPR/ENDOR studies (3).
References:
(1) Dubrovay Z et al (2004) Multidimensional NMR identifies the conformational shift essential for catalytic competence in the 60 kDA Drosophila melanogaster dUTPase trimer.
J Biol Chem, 279, 17945-50.
(2) Kovári J et al. (2004) Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase.
J Biol Chem, 279, 17932-44..
(3) Mustafi D, Békési A, Vártessy, BG*, Makinen, MW* (*corresponding authors) (2003) The catalytic and structural role of the metal ion in dUTP pyrophosphatase Proc Natl Acad Sci U S A 100, 5670-5675.
Supported by HHMI #55000342, OTKA T 034120, Alexander von Humboldt Foundation
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