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Peptide Deformylase (def) is essential in Mycobacterium smegmatis, but the essentiality is compensated by inactivation of methionine formylation.

ABSTRACT: BACKGROUND:Co-translational processes in bacteria are attractive drug targets, but while some processes are essential, others are not. The essentiality of Peptide Deformylase (PDF, def) for vitality of mycobacteria was speculated, but never unequivocally proven. RESULTS:Here we show by targeted deletion experiments that def can only be deleted from M. smegmatis when an additional copy is present; that prior deletion of tRNAfMet-Formyl Transferase (FMT, encoded by fmt) renders def completely dispensable; and that re-introduction of fmt into a ?def mutant is not possible - constituting a definitive proof for the essentiality of def in mycobacteria. CONCLUSIONS:Peptide deformylase is essential in M. smegmatis, but the fact that inactivation of fmt renders the gene completely dispensable, and thus any inhibitor of def useless, casts doubt on the usefulness of PDF as a drug-target in mycobacteria.

SUBMITTER: Naor N

PROVIDER: S-EPMC6815462 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Peptide Deformylase (def) is essential in Mycobacterium smegmatis, but the essentiality is compensated by inactivation of methionine formylation.

Naor Noga N Gadot Omer O Meir Michal M Barkan Daniel D

BMC microbiology 20191026 1

<h4>Background</h4>Co-translational processes in bacteria are attractive drug targets, but while some processes are essential, others are not. The essentiality of Peptide Deformylase (PDF, def) for vitality of mycobacteria was speculated, but never unequivocally proven.<h4>Results</h4>Here we show by targeted deletion experiments that def can only be deleted from M. smegmatis when an additional copy is present; that prior deletion of tRNA<sup>fMet</sup>-Formyl Transferase (FMT, encoded by fmt) r ...[more]

PMID: 31655553

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Project description:BackgroundTuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.ResultsThe gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM.ConclusionThe methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.

Project description:UnlabelledSuccinate: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 mc(2)155 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 ?sdh1 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.ImportanceComplex 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.

Dataset Information

Peptide Deformylase (def) is essential in Mycobacterium smegmatis, but the essentiality is compensated by inactivation of methionine formylation.

Publications

Peptide Deformylase (def) is essential in Mycobacterium smegmatis, but the essentiality is compensated by inactivation of methionine formylation.

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