Homotrimeric dUTPases; structural solutions for specific recognition and hydrolysis of dUTP.

Abstract

Prevention of incorporation of dUTP into DNA is essential for maintenance of the genetic information. Prompt and specific removal of dUTP from the nucleotide pool, as expedited by the ubiquitous enzyme dUTPase, is therefore required for full viability in most biological systems. Conserved structural features perpetuate specificity in choice of substrate, which is crucial as hydrolysis of the structurally closely related nucleotides dTTP, dCTP and UTP would debilitate DNA and RNA synthesis. The most common family of dUTPases is the homotrimeric variety where X-ray structures are available for one bacterial, one mammalian and two retroviral dUTPases. These four enzymes have similar overall structural layouts, but the interactions that stabilise the trimer vary markedly, ranging from exclusively hydrophobic to water-mediated interactions. Trimeric dUTPases contain five conserved sequence motifs, positioned at the subunit interfaces where they contribute to the formation of the active sites. Each of the three identical active sites per trimer is built of residues contributed by all three subunits. One subunit provides residues involved in base and sugar recognition, where a beta-hairpin acts to maintain exquisite selectivity, while a second subunit contributes residues for phosphate interactions. The third subunit supplies a glycine-rich consensus motif located in the flexible C-terminal part of the subunit, known from crystallographic studies to cover the active site in the presence of substrate and certain substrate analogues. All dUTPases studied require the presence of a divalent metal ion, preferably Mg(2+), for optimal activity. The putative position of the essential metal ion has been identified in the structure of one retroviral dUTPase. Structure-function studies are essential if the properties of dUTPases are to be understood fully in relation to their biological role. In this review the structural arrangement of the homotrimeric dUTPases is discussed in the context of active site geometry, achievement of specificity and subunit interactions.