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Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action

Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNA(fMet) to fMet-tRNA(fMet) is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene t...

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Autores principales: Sah, Shivjee, Varshney, Umesh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Microbiology Society 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10197868/
https://www.ncbi.nlm.nih.gov/pubmed/36745551
http://dx.doi.org/10.1099/mic.0.001297
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author Sah, Shivjee
Varshney, Umesh
author_facet Sah, Shivjee
Varshney, Umesh
author_sort Sah, Shivjee
collection PubMed
description Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNA(fMet) to fMet-tRNA(fMet) is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH(2)-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNA(fMet). Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH(2)-THF, 5-CH(3)-THF, 5,10-CH(+)-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNA(fMet).
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spelling pubmed-101978682023-05-20 Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action Sah, Shivjee Varshney, Umesh Microbiology (Reading) Microbial Physiology, Biochemistry and Metabolism (formerly Physiology and Metabolism) Methionyl-tRNA formyltransferase (Fmt)-mediated formylation of Met-tRNA(fMet) to fMet-tRNA(fMet) is crucial for efficient initiation of translation in bacteria and the eukaryotic organelles. Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, carries out conversion of 5,10-methylene tetrahydrofolate (5,10-CH(2)-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using in vivo and in vitro approaches, we show that 10-CHO-DHF may also be utilized by Fmt as an alternative substrate (formyl group donor) to formylate Met-tRNA(fMet). Dihydrofolate (DHF) formed as a by-product in the in vitro assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expressing strains were more sensitive to trimethoprim (TMP) than the ∆fmt strain, suggesting that the domino effect of TMP leads to inhibition of protein synthesis and strain growth. Antifolate treatment to Escherichia coli showed a decrease in the reduced folate species (THF, 5,10-CH(2)-THF, 5-CH(3)-THF, 5,10-CH(+)-THF and 5-CHO-THF) and increase in the oxidized folate species (folic acid and DHF). In cells, 10-CHO-DHF and 10-CHO-folic acid were enriched in the stationary phase. This suggests that 10-CHO-DHF is a bioactive metabolite in the folate pathway for generating other folate intermediates and fMet-tRNA(fMet). Microbiology Society 2023-02-06 /pmc/articles/PMC10197868/ /pubmed/36745551 http://dx.doi.org/10.1099/mic.0.001297 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License.
spellingShingle Microbial Physiology, Biochemistry and Metabolism (formerly Physiology and Metabolism)
Sah, Shivjee
Varshney, Umesh
Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
title Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
title_full Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
title_fullStr Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
title_full_unstemmed Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
title_short Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
title_sort methionyl-trna formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action
topic Microbial Physiology, Biochemistry and Metabolism (formerly Physiology and Metabolism)
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10197868/
https://www.ncbi.nlm.nih.gov/pubmed/36745551
http://dx.doi.org/10.1099/mic.0.001297
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