The dUTPase Enzyme Is Essential in Mycobacterium smegmatis

Abstract

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. All mycobacterial genomes encode dUTPase with a mycobacteria-specific surface loop absent in the human dUTPase. Using Mycobacterium smegmatis as a fast growing model for Mycobacterium tuberculosis, we demonstrate that dUTPase knock-out results in lethality that can be reverted by complementation with wild-type dUTPase. Interestingly, a mutant dUTPase gene lacking the genus-specific loop was unable to complement the knock-out phenotype. We also show that deletion of the mycobacteria-specific loop has no major effect on dUTPase enzymatic properties in vitro and thus a yet to be identified loop-specific function seems to be essential within the bacterial cell context. In addition, here we demonstrated that Mycobacterium tuberculosis dUTPase is fully functional in Mycobacterium smegmatis as it rescues the lethal knock-out phenotype. Our results indicate the potential of dUTPase as a target for antitubercular drugs and identify a genus-specific surface loop on the enzyme as a selective target.

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

“…dCTP and dTTP). In a recent study, we showed that a mycobacterium specific structural motif is essential for viability potentially due to a yet unidentified protein-protein interaction [23]. This putative interaction was most likely not perturbed by Stl, as the bacteria expressing the exogenous inhibitor protein remained viable, although colony formation was decreased.…”

“…This enzyme shows a very similar homotrimeric fold and catalyses both the dCTP deamination reaction and the triphosphate hydrolysis of the resulting dUTP, directly producing dUMP from dCTP. However, the efficiency of the triphosphate hydrolysis by the bifunctional enzyme is several hundred fold less [19] than that of the monofunctional trimeric dUTPase [20].The monofunctional dUTPase (dut) is essential in Mycobacteria [21][22][23]. In contrast, earlier mutagenesis studies found that the presence of the bifunctional dCTP deaminase:dUTPase enzyme is dispensable for growth in M. tuberculosis [21,22].…”

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes

“…We carried out the further detailed biochemical and structural biology experiments to validate this in silico observation and to learn more about the substrate binding process. (33,42,49). Position 21 is conserved in the bacterial phylum Actinobacteria.…”

“…These results obtained from crystal structures and from spectroscopic measurements on the millisecond time scale movements did not allow insights into the possible substrate binding pathways to the hidden active site of dUTPase. This process may have pharmacological significance because we have recently shown that the dUTPase enzyme may be a selective drug target in mycobacteria (42). This enzyme plays a crucial role in DNA integrity by catalyzing the hydrolysis of dUTP into dUMP and pyrophosphate, producing the precursor for dTTP biosynthesis.…”

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

“…The interest in these enzymes is highly motivated on the one hand by the peculiar active site architecture and, on the other hand, by the possibility of using these dUTPases as targets for developing drugs against neoplastic, as well as infectious diseases [31,[56][57][58][59][60][61][62][63][64][65] [100]. All-b dUTPases also include monomeric dUTPases [40].…”