Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids

Abstract

We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels. The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate. The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates. The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP. The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i). dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF. In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function.

On the catalytic mechanism of dimeric dUTPases

“…The present TbdUTPase dUpNp complex structures both have Ca 2 + ions in sites 1 and 2 (Supplementary Figures S3A and S3B at http://www.biochemj.org/bj/456/bj4560081add.htm). The metals are absolutely required for activity [20], whereas the trimeric enzyme can hydrolyse dUTP in the absence of Mg 2 + , albeit at a much lower rate [48]. Similarly, for the [AlF 3 -OPO 3 ] complex, Mg 2 + was present in sites 1 and 2 with Na + in site 3, whereas with MgF 3 − the three sites were all occupied by Mg 2 + (Supplementary Figures S3C and S3D).…”

“…Further to their protective role, secondary 'moonlighting' functions have been proposed for viral/phage dUTPases, suggesting that they may also have a signalling role similar to that of small GTPases [10][11][12][13][14][15]. Furthermore, the Tritryp enzymes are subject to product inhibition, the trimeric form much less so [20]. These trimeric enzymes are found in most organisms including humans, with structures consisting largely of β-sheets [16].…”

Fluoride and organic weak acids as modulators of microbial physiology

“…Fluoride can affect a variety of phosphatases. For example, Camacho et al [20] found that, in contrast to most dUTP nucleohydrolases studied previously, the enzyme of kinetoplasts of Leishmania is remarkably sensitive to NaF and could be inhibited 73% by 0.2 mM fluoride. There was no stated need for co‐ions such as aluminum, and inhibition may have been due simply to binding of fluoride, especially in view of the activation of the enzyme by Mg 2+ or Mn 2+ .…”

Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease

“…The last subgroup is the homodimeric dUTPases, which were identified in Leshmania major [8,9], Trypansoma cruzi [10], Caenorhabditis elegans [11], and Campylobacter jejuni [12]. The homodimeric dUTPases differ not only structurally from the monomeric and homotrimeric dUTPases, but they also exhibit broader substrate specificity.…”

“…The homodimeric dUTPases differ not only structurally from the monomeric and homotrimeric dUTPases, but they also exhibit broader substrate specificity. Furthermore, sequence comparisons have demonstrated that the dimeric dUTPases lack the five consensus amino acid motifs found in mono- and trimeric dUTPases and they are evolutionary related to the dCTPase-dUTPase of bacteriophages T2 and T4 [9]. Protein sequence alignments of the herpesviruses and human nuclear dUTPases are shown in Figure 1.…”