Formate-tetrahydrofolate ligase: supplying the cytosolic one-carbon network in roots with one-carbon units...

The metabolism of tetrahydrofolate (H₄PteGlu_n)-bound one-carbon (C₁) units (C₁ metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C₁ units in specific organelles and tissues. One possible source of C₁ units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H₄PteGlu_n) from formate and tetrahydrofolate (H₄PteGlu_n). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower K_m and k_cat values with pentaglutamyl tetrahydrofolate (H₄PteGlu₅) as compared to monoglutamyl tetrahydrofolate (H₄PteGlu₁), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H₄PteGlu_n + 5,10-methylene-H₄PteGlu_n and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C₁ network in roots with C₁ units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H₄PteGlu_n, methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs.