The dUTPase enzyme is essential in Mycobacterium smegmatis.
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.
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest, Hungary.
Ildiko
I
Pecsi
Rita
R
Hirmondo
Amanda C
AC
Brown
Anna
A
Lopata
Tanya
T
Parish
Beata G
BG
Vertessy
Judit
J
Tóth
D000595:Amino Acid Sequence; D055786:Gene Knockout Techniques; D023281:Genomics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009165:Mycobacterium Infections, Nontuberculous / Q000201:enzymology / Q000821:virology*; D020102:Mycobacterium smegmatis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase