Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

Abstract

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.

GTP Is the Primary Activator of the Anti-HIV Restriction Factor SAMHD1

“…dUTP is a non-canonical nucleotide formed in the cell as a side product of dTTP formation. dUTPase is an essential enzyme expressed by the cell to decrease the concentration of dUTP, thus preventing incorporation of dUMP into genomic DNA (19). This result suggests that SAMHD1 also contributes to maintenance of the cellular dUTP concentration.…”

Exploring Bacterial Carboxylate Reductases for the Reduction of Bifunctional Carboxylic Acids

“…The reaction products (ADP and ATP) were analyzed using reversed phase chromatography on a Varian ProStar HPLC system equipped with a Varian Pursuit C18 column as previously described [37, 38]. Standard solutions of AMP, ADP, and ATP were used to confirm the identity of the observed peaks and products.…”

Tying down the arm inBacillusdUTPase: structure and mechanism

“…2c) to those observed in form I, with the only difference being the manner in which the cubes pack against one another. Furthermore, in the 24-mer asymmetric unit (in space group P1) of yeast apo dUTPase (Tchigvintsev et al, 2011) we identified a similar cube but again with different packing between adjacent cubes within the crystal lattice. The yeast dUTPase cube does not superpose exactly on those of the B. subtilis dUTPases, reflecting some distortion of the intra-cube molecular packing (Fig.…”

“…Four of the motifs are contributed by two adjacent subunits, while motif V, which is located in a flexible C-terminal arm, originates from the third subunit and caps the active site, alternating between closed and open states along the reaction cycle. This C-terminal arm is disordered in most of the published structures, only adopting an ordered conformation in M. tuberculosis dUTPase in complex with Mg 2+ -dUpNHpp , FIV dUTPase in complex with dUDP (Prasad et al, 2000), the human dUTPase complexes with dUMP, dUDP and dUTP and a recent structure of the yeast dUTPasedUMP complex (Tchigvintsev et al, 2011). Motif V is the least understood motif and has been the subject of several recent studies (Pé csi et Bacillus subtilis is a model organism for Gram-positive bacteria and plays a role in industrial processes through its ability to produce and secrete large amounts of proteins such as amylases and proteases (Harwood, 1992).…”