biostudies-literatureUnknown169(2)Sah SScience and Education Research Board, New Delhi, IndiaTata Trusts (IISc), IndiaDepartment of Biotechnology, Ministry of Science and Technology, IndiaMethionyl-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-CH₂-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using <i>in vivo</i> and <i>in vitro</i> 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 <i>in vitro</i> assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expreMicrobiology (Reading, England)https://www.ebi.ac.uk/biostudies/studies/S-EPMC10197868biostudies-literatureMethionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action.PMC10197868Sah SVarshney UfalseMethionyl-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 tetrahydrofolate (5,10-CH₂-THF) to 10-formyl-THF (10-CHO-THF), a metabolite utilized by Fmt as a formyl group donor. In this study, using <i>in vivo</i> and <i>in vitro</i> 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 <i>in vitro</i> assay was verified by LC-MS/MS analysis. FolD-deficient mutants and Fmt over-expre2023-01-01T00:00:00Z2023 Feb2025-04-04T23:44:39.959Z2025-02-19T03:55:29.849ZS-EPMC101978683674555110.1099/mic.0.001297