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

A hidden active site in the potential drug target Mycobacterium tuberculosis dUTPase is accessible through small-amplitude protein conformational changes

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent

Beyond chelation:EDTA tightly binds taq DNA polymerase, MutT and dUTPase and directly inhibits dNTPase activity

Abstract EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments