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

Sequence and structural comparison of selected members of the dUTPase superfamily.

<p>(<b>A</b>) Conserved motifs are indicated above the sequences as lines. Representative organisms from widely different evolutionary branches are also included for comparison. Mycobacterial dCTP deaminases contain all those conserved residues that are indispensable for dUTPase reaction. Residues conserved between dUTPases and bifunctional dCTP deaminase/dUTPases are important for the dephosphorylation reaction and indicated with green boxes. Residues important for the deamination reaction and crucial for dCTP deaminase monofunctionality are depicted as gray and magenta boxes, respectively. Mycobacterial dUTPases contain an insert present solely in the mycobacterial <i>dut</i>, this insert is shown as a yellow box. The alignment was performed with ClustalW. (<b>B</b>) The mycobacterial insert induces a loop structure on the surface of the dUTPase monomer. The superimposed structure of hDUT (PDB ID: 3EHW, under publication in a separate paper) and mtDUT (PDB ID: 2PY4) are depicted as yellow and green cartoon representation, respectively. The mycobacterial insert can be seen as cartoon tube representation. In the active sites the bound ligands, dUPNPP and Mg²⁺ can be seen, whereby the Mg²⁺ is visualized as a yellow (hDUT) or green (mtDUT) sphere while dUPNPP is represented as sticks with atomic coloring (carbons in yellow and green). Structures were prepared using PyMol. (<b>C</b>) Superimposed overall structure of the <i>M. tuberculosis</i> bifunctional dCTP deaminase/dUTPase and the <i>M. smegmatis</i> dCTP deaminase enzymes in green and magenta cartoon representation, respectively. (<b>D</b>) Enlarged view from <b>C</b> showing the conserved residues and the non-hydrolysable substrate analog α-β-imido-dUTP (dUPNPP) as modeled to the active site. Residues and the dUPNPP molecule are in stick representation with atomic coloring (green, magenta and cyan carbons for <i>M. tuberculosis</i>, <i>M. smegmatis</i> enzymes and dUPNPP, respectively). Note the closely identical organization of both the overall structure and the active site in <i>M. tuberculosis</i> and <i>M. smegmatis</i> dUTPases.</p

Homology of <i>M. tuberculosis</i> and <i>M. smegmatis</i> proteins present in the thymidylate synthesis pathway.

<p>1 =  % identical amino-acids;</p><p>2 =  classified on the basis of chemical properties (e.g. polar vs. non-polar) of the respective amino-acids side chains.</p

The Δ-loop mutant dUTPase is unable to rescue the lethal phenotype despite its normal expression level.

<p>(<b>A</b>) Colony PCR analysis of the generated double crossover (DCO) strains. 88 strains were screened and no mutant cell line could be isolated. For demonstration, only a subset of the samples are shown here. M stands for the 1 kb DNA marker from Fermentas. The identical numbers represent samples from the same cell line. Every cell line was screened for both the WT copy (indicated as 1, 2, 3, 4) and for the disrupted mutant <i>dut</i> gene (labeled as 1′ 2′ 3′ 4′). The lengths of the expected PCR product for the wild type (WT) <i>dut</i> gene and for the disrupted <i>dut</i> mutant were 0.7 and 1.1 kb, respectively. (<b>B</b>) Western-blot analysis of FLAG-tagged WT and Δ-loop dUTPase expression in <i>M. smegmatis</i> transformed with the appropriate construct.</p

Schematic representation of allelic replacement by homologous recombination

<p>. (<b>A</b>) Generation of SCO strains. p2Nbk-<i>dut</i>h was electroporated into WT competent <i>M. smegmatis</i>, and single-crossover (SCO) transformants were selected. (<b>B</b>) Merodiploid strains were constructed by electroporating the complementing plasmid (pGem-<i>dut</i>) into the SCO strains. (<b>C</b>) Generation of disrupted <i>dut</i> deletion mutant strain. The double crossover event may result either a disrupted <i>dut</i> deletion mutant strain (a), or a wild type strain (b). (<b>D</b>) Strategy for SCO and DCO screening. a) shows primers and expected PCR products for the knock-out (KO) allele while b) shows the same for the WT allele. Abbreviations: WT; wild type; SCO; single crossover; DCO; double cross over.</p

Genomic environment of the <i>dut</i> gene.

<p>Arrangement of the neighboring genes on the chromosome of the mc² -155 <i>M. smegmatis</i> strain is shown together with the regions amplified for the construction of p2Nbk-<i>dut</i>h and pGem-<i>dut</i>. Relevant restriction sites are also shown. The chromosomal location of the <i>dut</i> gene is represented by black arrow, the region cloned into the delivery vector (p2Nbk-<i>dut</i>h) is indicated with a black rectangle, and the region cloned in the complementing vector (pGem-<i>dut</i>) is shown by a white rectangle.</p

Effect of the Δ-loop mutation on the substrate hydrolysis and binding of <i>M. tuberculosis</i> dUTPase.

<p> (<b>A</b>) SDS-PAGE analysis of the purified proteins used in this study. M stands for the PageRuler Plus Prestained Protein Ladder (Fermentas). The WT and Δ-loop mutant dUTPases have calculated molecular weights of 18.0 kDa and 17.6 kDa, respectively. (<b>B</b>) The steady-state activity of WT and Δ-loop mutant dUTPase is shown. Michaelis-Menten curves for the WT (squares) and the Δ-loop mutant (triangle) were measured using the phenol red pH indicator assay. Fitting the Michaelis-Menten equation to the curves yielded the following V_max and K_M values: 1.22±0.06 s⁻¹ and 0.9±0.5 μM for WT, 0.88±0.02 s⁻¹ and 1.1±0.2 μM for Δ-loop. (<b>C</b>) Fluorescence intensity titration of the WT and the Δ-loop mutant using the single Trp signal is shown upon dUPNPP binding. Smooth lines through the data are quadratic fits yielding the K_d values listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037461#pone-0037461-t002" target="_blank">Table 2</a>. Errors represent S.D. for n = 3. For more parameters see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037461#pone-0037461-t002" target="_blank">Table 2</a>. (<b>D</b>) CD equilibrium titrations. Comparison of ligand (dUPNPP) binding to the WT and to the Δ-loop mutant dUTPase. Smooth lines represent quadratic fits to the data yielding the following K_d values: 0.9±0.5 μM for WT and 3.9±2.4 μM for Δ-loop.</p

The <i>dut</i> gene is essential in <i>M. smegmatis</i>.

<p>M stands for the 1 kb DNA marker from Fermentas. (<b>A</b>) Identification of SCO strain by colony PCR. SCO strains were generated by homolog recombination of p2Nbk-<i>dut</i>h with chromosomal copy of <i>dut</i>. Chromosomal DNA from <i>M. smegmatis</i> mc ²-155 was used as a positive control yielding the 486 bp fragment (lane WT); the suicide vector integration due to single-crossover event yielded the 860 bp fragment. (lane SCO). (<b>B</b>) Colony PCR analysis of the generated double crossover (DCO) strains. For demonstration, only a subset of 19 samples are shown here. The identical numbers represent samples from the same cell line. The potential DCO cell lines were screened for both the WT copy (indicated as 2, 3) and for the disrupted deletion mutant <i>dut</i> gene (labeled as 2′ 3′). The lengths of the expected PCR product for the wild type (WT) <i>dut</i> gene and for the disrupted <i>dut</i> mutant were 0.7 and 1.1 kb, respectively. (<b>C</b>) Southern blot analysis of DCOs. The probe used to perform the hybridization corresponds to the 1.5 kb WT (lane 1) and the 3.3 kb disrupted <i>dut</i> deletion mutant (lane 2 and 3) restriction fragment, respectively.</p

Kinetic parameters of WT and Δ-loop deletion mutant <i>M. tuberculosis</i> dUTPase enzymes and dissociation constants of dUTPase-dUPNPP complexes.

<p>Kinetic parameters of WT and Δ-loop deletion mutant <i>M. tuberculosis</i> dUTPase enzymes and dissociation constants of dUTPase-dUPNPP complexes.</p

Key enzymes of the <i>de novo</i> thymidylate biosynthesis pathway in mycobacteria.

<p>Various enzymes present in this pathway are as follows: bifunctional deoxycytidine triphosphate deaminase/ deoxyuridine triphosphate nucleotidohydrolase (bifunctional dCTPdeaminase/ dUTPase), deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase), nucleoside diphosphate kinase (Ndk), thymidylate kinase (dTMP kinase), thymidylate synthase (ThyA, ThyX) and ribonucleoside diphosphate reductase (Nrd). The dUTPase enzyme (underlined) converts dUTP (grey highlighted box) into dUMP (grey highlighted box) thereby provides input into dTTP synthesis and eliminates dUTP. An abnormally elevated dUTP/dTTP ratio will lead to uracil incorporation into DNA, as indicated by the dashed arrow. DNA synthesis is provided by several different polymerases (for simplicity no specific polymerases are named here).</p