title: 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.
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 ((31)P-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.
authors: Laboratory of Genome Metabolism, Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest H-1113, Hungary, Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD 20892, USA, Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg D-69117, Germany, Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Budapest H-1117, Hungary, Protein Modelling Group MTA-ELTE, Institute of Chemistry, Eötvös Loránd University, Budapest H-1117, Hungary, Department of Chemistry, King's College London, London, SE1 1UL, UK, Department of Medical Chemistry, University of Szeged, Hungary, Department of Computer Science, Eötvös Loránd University, Budapest, Hungary, European Molecular Biology Laboratory, Hamburg Outstation, Hamburg D-22603, Germany and Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, Hungary.
authors: Orsolya
authors: O
authors: Barabás
authors: Veronika
authors: V
authors: Németh
authors: Andrea
authors: A
authors: Bodor
authors: András
authors: A
authors: Perczel
authors: Edina
authors: E
authors: Rosta
authors: Zoltán
authors: Z
authors: Kele
authors: Imre
authors: I
authors: Zagyva
authors: Zoltán
authors: Z
authors: Szabadka
authors: Vince I
authors: VI
authors: Grolmusz
authors: Matthias
authors: M
authors: Wilmanns
authors: Beáta G
authors: BG
authors: Vértessy
mesh_terms: D055162:Biocatalysis; D018360:Crystallography, X-Ray; D003856:Deoxyuracil Nucleotides / Q000737:chemistry / Q000378:metabolism; D016093:Mason-Pfizer monkey virus / Q000201:enzymology; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010710:Phosphates / Q000737:chemistry*; D010759:Phosphorus Isotopes; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism
chemical_list: C110398:2'-deoxyuridine 5'-imidotriphosphate; D003856:Deoxyuracil Nucleotides; D010710:Phosphates; D010759:Phosphorus Isotopes; C027078:deoxyuridine triphosphate; C007267:2'-deoxyuridylic acid; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase
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