Structure and enzymatic mechanism of a moonlighting dUTPase

Abstract

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. In Staphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Å resolution three-dimensional structure of a ϕ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small β-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of ϕ11 phage dUTPase.

The Type 2 dUTPase of Bacteriophage ϕNM1 Initiates Mobilization of Staphylococcus aureus Bovine Pathogenicity Island 1

“…4C), comparable to other members of the dimeric, α-helical pyrophosphohydrolase superfamily [31]. By comparison, the type 1 Dut 11 hydrolyzed dUTP with a V max =0.44μM/s, K M =1.2 μM [22, 27]. …”

“…Type 1 dUTPases, the most common class, consist of an antiparallel β-sandwich and are usually trimeric [28–30]. The Dut proteins of 80α and the closely related phage ϕ11 belong to this class [22, 27] (Supplementary Fig. S1A).…”

“…Here, we show that ORF31 encodes a dUTPase (Dut NM1 ) that belongs to the dimeric α-helical family of dUTPases, whereas Dut 80α and Dut 11 have a trimeric β-sheet structure [22, 27]. Further, we show that no other ϕNM1-encoded protein is involved in the derepression of SaPIbov1, and that there is a direct interaction between SaPIbov1 Stl and Dut NM1 .…”

Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP

“…The helper phages initiate SaPI mobilization by producing proteins that interact with the SaPI master repressor, Stl, leading to derepression and expression of genes involved in SaPI excision and replication [19]. SaPIbov1 was shown to be derepressed by the type 1 dUTPase encoded on phages 80α and φ11 (Dut 80α and Dut 11 respectively) [19, 20]. We showed previously that SaPIbov1 could also be derepressed by the type 2 dUTPase from phage φNM1 (Dut NM1 ) [21, 22].…”

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

“…These results, combined with phylogenetic analysis, allowed the definition of a new superfamily of d(C/U)TPases [36], including several distinct enzyme families (dUTPases in trypanosomatides, Campylobacter jejuni and dCTP/dUTPases in some Gram-negative bacteria and T4-like phages, as well as dUTPases in various Gram-positive bacteria and their phages) [89][90][91]. This branch of dUTPase enzymes was later confirmed to belong to the all-a NTP pyrophosphohydrolase superfamily, possessing house-cleaning functions [92].…”