Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling

Orsolya Barabás, Veronika Németh, A. Bodor, A. Perczel, Edina Rosta, Z. Kele, Imre Zagyva, Zoltán Szabadka, Vince I. Grolmusz, Matthias Wilmanns, B. Vértessy

Research output: Contribution to journalArticle

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Abstract

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the α-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue α,β-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure.

Original languageEnglish
Pages (from-to)10542-10555
Number of pages14
JournalNucleic Acids Research
Volume41
Issue number22
DOIs
Publication statusPublished - Dec 2013

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Magnetic Resonance Spectroscopy
Phosphates
X-Rays
Mechanics
Nucleosides
Simian Retroviruses
Hydrolysis
DNA
Phosphorus
Signal Transduction
Mass Spectrometry
dUTP pyrophosphatase
Enzymes

ASJC Scopus subject areas

  • Genetics

Cite this

Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling. / Barabás, Orsolya; Németh, Veronika; Bodor, A.; Perczel, A.; Rosta, Edina; Kele, Z.; Zagyva, Imre; Szabadka, Zoltán; Grolmusz, Vince I.; Wilmanns, Matthias; Vértessy, B.

In: Nucleic Acids Research, Vol. 41, No. 22, 12.2013, p. 10542-10555.

Research output: Contribution to journalArticle

Barabás, Orsolya ; Németh, Veronika ; Bodor, A. ; Perczel, A. ; Rosta, Edina ; Kele, Z. ; Zagyva, Imre ; Szabadka, Zoltán ; Grolmusz, Vince I. ; Wilmanns, Matthias ; Vértessy, B. / Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling. In: Nucleic Acids Research. 2013 ; Vol. 41, No. 22. pp. 10542-10555.
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