18 research outputs found
The dUTPase Enzyme Is Essential in Mycobacterium smegmatis
No full textPubMed ID: 22655049This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Essentiality of Succinate Dehydrogenase in <named-content content-type="genus-species">Mycobacterium smegmatis</named-content> and Its Role in the Generation of the Membrane Potential Under Hypoxia
No full textABSTRACT Succinate:quinone oxidoreductase (Sdh) is a membrane-bound complex that couples the oxidation of succinate to fumarate in the cytoplasm to the reduction of quinone to quinol in the membrane. Mycobacterial species harbor genes for two putative sdh operons, but the individual roles of these two operons are unknown. In this communication, we show that Mycobacterium smegmatis mc2155 expresses two succinate dehydrogenases designated Sdh1 and Sdh2. Sdh1 is encoded by a five-gene operon (MSMEG_0416-MSMEG_0420), and Sdh2 is encoded by a four-gene operon (MSMEG_1672-MSMEG_1669). These two operons are differentially expressed in response to carbon limitation, hypoxia, and fumarate, as monitored by sdh promoter-lacZ fusions. While deletion of the sdh1 operon did not yield any growth phenotypes on succinate or other nonfermentable carbon sources, the sdh2 operon could be deleted only in a merodiploid background, demonstrating that Sdh2 is essential for growth. Sdh activity and succinate-dependent proton pumping were detected in cells grown aerobically, as well as under hypoxia. Fumarate reductase activity was absent under these conditions, indicating that neither Sdh1 nor Sdh2 could catalyze the reverse reaction. Sdh activity was inhibited by the Sdh inhibitor 3-nitroproprionate (3NP), and treatment with 3NP dissipated the membrane potential of wild-type or Δsdh1 mutant cells under hypoxia but not that of cells grown aerobically. These data imply that Sdh2 is the generator of the membrane potential under hypoxia, an essential role for the cell. IMPORTANCE Complex II or succinate dehydrogenase (Sdh) is a major respiratory enzyme that couples the oxidation of succinate to fumarate in the cytoplasm to the reduction of quinone to quinol in the membrane. Mycobacterial species harbor genes for two putative sdh operons, sdh1 and sdh2, but the individual roles of these two operons are unknown. In this communication, we show that sdh1 and sdh2 are differentially expressed in response to energy limitation, oxygen tension, and alternative electron acceptor availability, suggesting distinct functional cellular roles. Sdh2 was essential for growth and generation of the membrane potential in hypoxic cells. Given the essentiality of succinate dehydrogenase and oxidative phosphorylation in the growth cycle of Mycobacterium tuberculosis, the potential exists to develop new antituberculosis agents against the mycobacterial succinate dehydrogenase. This enzyme has been proposed as a potential target for the development of new chemotherapeutic agents against intracellular parasites and mitochondrion-associated disease
Schematic representation of allelic replacement by homologous recombination
No full text<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
Key enzymes of the <i>de novo</i> thymidylate biosynthesis pathway in mycobacteria.
No full text<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
Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase.
No full textAromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π-π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis
Kinetic parameters of WT and Δ-loop deletion mutant <i>M. tuberculosis</i> dUTPase enzymes and dissociation constants of dUTPase-dUPNPP complexes.
No full text<p>Kinetic parameters of WT and Δ-loop deletion mutant <i>M. tuberculosis</i> dUTPase enzymes and dissociation constants of dUTPase-dUPNPP complexes.</p
Homology of <i>M. tuberculosis</i> and <i>M. smegmatis</i> proteins present in the thymidylate synthesis pathway.
No full text<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 <i>M. tuberculosis</i> dUTPase is able to complement the lethal phenotype in <i>M. smegmatis</i>
No full text<p>. (<b>A</b>) Colony PCR analysis of the generated DCO strains. For demonstration, only a subset of 20 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) and for the disrupted deletion mutant <i>dut</i> gene (labeled as 1′ 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>B</b>) Southern-blot analysis of <i>dut</i> disrupted, <i>M. tuberculosis dut</i> coding mutants. WT was used for control. The probe used to perform the hybridization corresponds to the 1.5 kb WT (lane 1) and the 3.3 kb <i>dut</i> disrupted mutant (lane 2 and 3) restriction fragment, respectively. (<b>C</b>) Western-blot analysis of FLAG-tagged <i>M. tuberculosis</i> dUTPase expression in <i>M. smegmatis.</i></p
Effect of the Δ-loop mutation on the substrate hydrolysis and binding of <i>M. tuberculosis</i> dUTPase.
No full text<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<sub>max</sub> and K<sub>M</sub> values: 1.22±0.06 s<sup>−1</sup> and 0.9±0.5 μM for WT, 0.88±0.02 s<sup>−1</sup> 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<sub>d</sub> 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<sub>d</sub> values: 0.9±0.5 μM for WT and 3.9±2.4 μM for Δ-loop.</p
