dUTPase based switch controls transfer of virulence genes in order to preserve integrity of the transferred mobile genetic elements

dUTPases ubiquitously regulate cellular dUTP levels to preserve genome integrity. Recently, several other cellular processes were reported to be controlled by dUTPases including the horizontal transfer of Staphylococcus aureus pathogenicity islands (SaPI). SaPIs are mobil genetic elements that encode virulence enhancing factors e.g. toxins. Here, phage dUTPases were proposed to counteract the repressor protein (Stl) and promote SaPI excision and transfer. A G protein-like mechanism was proposed which is unexpected in light of the kinetic mechanism of dUTPase. Here we investigate the molecular mechanism of SaPI transfer regulation, using numerous dUTPase variants and a wide range of in vitro methods (steady-state and transient kinetics, VIS and fluorescence spectroscopy, EMSA, quartz crystal microbalance, X-ray crystallography). Our results unambiguously show that Stl inhibits the enzymatic activity of dUTPase in the nM concentration range and dUTP strongly inhibits the dUTPase: Stl complexation. These results identify Stl as a highly potent dUTPase inhibitor protein and disprove the G protein-like mechanism. Importantly, our results clearly show that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Unlike in small GTPases, hydrolysis of the substrate nucleoside triphosphate (dUTP in this case) is required prior to the interaction with the partner (Stl repressor in this case). We propose that dUTPase can efficiently interact with Stl and induce SaPI excision only if the cellular dUTP level is low (i.e. when dUTPase resides mainly in the apo enzyme form) while high dUTP levels would inhibit SaPI transfer. This mechanism may serve the preservation of the integrity of the transferred SaPI genes and links the well-known metabolic role of dUTPases to their newly revealed regulatory function in spread of virulence factors

Catalytic mechanism of alpha-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the alpha-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue alpha,beta-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure