Abstract
Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.
Highlights
Various members of the genus Leishmania cause leishmaniasis, which threatens ϳ350 million people worldwide and gives rise to about two million clinical cases each year, of which ϳ25% are of the fatal visceral form [1]
Inhibition of Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity results in futile cycles of DNA repair that cause the fragmentation of DNA and cell death [4, 5]. dUTPase activity appears to be essential in L. major [6] and the related parasite Trypanosoma brucei, where an RNAi approach was used to knock down dUTPase expression, resulting in decreased cell proliferation and growth in both procyclic and bloodstream forms of the organism [7]
The metals were implicated in substrate binding and led to the proposal that these dimeric dUTPases utilize a mechanism of reaction similar to that used in the phosphoryl transfer reactions catalyzed by DNA polymerases [20, 21]
Summary
Various members of the genus Leishmania cause leishmaniasis, which threatens ϳ350 million people worldwide and gives rise to about two million clinical cases each year, of which ϳ25% are of the fatal visceral form [1]. A structure of the closed form of the dimeric dUTPase from Campylobacter jejuni (CjdUTPase) was determined in the presence of the nonhydrolyzable substrate analogue dUpNp [19] This revealed the presence of three Mg2ϩ ions in the active site adjacent to the phosphate groups of the substrate. The essential nature of dUTPases and the marked differences between the two forms of the enzyme in different species make the dimeric dUTPases an attractive target for anti-parasitic drug development With this in mind, we have undertaken a structural characterization of the L. major dUTPase in complex with various substrate fragments to better understand the substrate binding determinants and the requirements to induce closure of this family of enzymes. We present crystal structures of the closed L. major dUTPase in the presence of the non-hydrolyzable substrate analogues ␣,-imino-deoxyuridine triphosphate (dUpNpp) and ␣,-imino-deoxyuridine diphosphate (dUpNp) and divalent metal ions supporting the proposed mechanism for these dimeric enzymes. We present the structure of the CjdUTPase in complex with dUpNpp with important implications for the metal-binding properties of this family of enzymes
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