Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution (1 Å=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.
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Research Article|
June 28 2011
Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme
Anatoli Tchigvintsev;
Anatoli Tchigvintsev
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Alexander U. Singer;
Alexander U. Singer
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Robert Flick;
Robert Flick
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Pierre Petit;
Pierre Petit
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Greg Brown;
Greg Brown
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Elena Evdokimova;
Elena Evdokimova
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Alexei Savchenko;
Alexei Savchenko
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
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Alexander F. Yakunin
Alexander F. Yakunin
1
1Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, Centre for Structural Proteomics in Toronto, University of Toronto, Toronto, Canada, M5G 1L6
1To whom correspondence should be addressed (email a.iakounine@utoronto.ca).
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Publisher: Portland Press Ltd
Received:
February 22 2011
Revision Received:
May 04 2011
Accepted:
May 06 2011
Accepted Manuscript online:
May 06 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 437 (2): 243–253.
Article history
Received:
February 22 2011
Revision Received:
May 04 2011
Accepted:
May 06 2011
Accepted Manuscript online:
May 06 2011
Citation
Anatoli Tchigvintsev, Alexander U. Singer, Robert Flick, Pierre Petit, Greg Brown, Elena Evdokimova, Alexei Savchenko, Alexander F. Yakunin; Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme. Biochem J 15 July 2011; 437 (2): 243–253. doi: https://doi.org/10.1042/BJ20110304
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