Methionyl-tRNA formyltransferase utilizes 10-formyldihydrofolate as an alternative substrate and impacts antifolate drug action. Issue 2 (6th February 2023)

• Record Type: Journal Article
• Authors: Sah, Shivjee; Varshney, Umesh
• Is Part Of: Microbiology. Volume 169:Issue 2(2023)
• Publication Date: 2023-02-06
• Subjects: 10-CHO-DHF -- 10-CHO-THF -- DHF -- Fmt -- folate pathway -- one-carbon metabolic pathway -- THF; Microbiology -- Periodicals; 579
• DOI: 10.1099/mic.0.001297
• Languages: English
• ISSNs: 1350-0872

Abstract

Abstract : 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 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-CH2 -THF, 5-CH3 -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 .