Human dUTP pyrophosphatase: Uracil recognition by a beta hairpin and active sites formed by three separate subunits

Background: The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis. A high cellular dTTP:dUTP ratio is essential to avoid uracil incorporation into DNA, which would lead to strand breaks and cell death. We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-containing deoxyribonucleotides, dUMP, dUDP and dUTP. Results: The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll beta barrel, with the C-terminal beta strands interchanged among the subunits. The structure is similar to that of the E. coli enzyme, despite low sequence homology between the two enzymes. The nucleotide complexes reveal a simple and elegant way for a beta hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel beta hairpin and hydrogen bonds entirely to main-chain atoms. This interaction mimics DNA base pairing, selecting uracil over cytosine and sterically precluding thymine and ribose binding, Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps the substrate-bound active site, causing total complementary enclosure of substrate. To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis. Conclusions: The dUTPase nucleotide-binding sites incorporate some features of other nucleotide-binding proteins and protein kinases, but seem distinct in sequence and architecture, The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolved from a motif of this kind. These uracil-beta-hairpin interactions are an obvious way for peptides to become early coenzymes in an RNA world, providing a plausible link to the protein-DNA world. Within the beta hairpin, there is a tyrosine corner motif that normally specifies beta-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the beta hairpin itself.