An Allele of Glutamate Formiminotransferase Triggers 5‐Methyl‐Tetrahydrofolate‐to‐MeFox Conversion and Facilitates Folate Biofortification in Maize

This work demonstrates the distinct roles of the glutamate formiminotransferase (GFT) enzyme in plants. The GFT triggers the conversion of 5‐methyl‐tetrahydrofolate to MeFox in plants, a process distinct from its role as a formiminotransferase in mammals. A key allelic variation in GFT, with the G‐allele linked to high folate levels in maize, highlights its significant impact on folate metabolism and breeding. Abstract Identifying genes involved in folate accumulation is critical for elucidating the regulatory mechanisms of folate metabolism and breeding folate‐rich crops. Here, a natural A‐to‐G variation at the 682nd bp is identified in the coding sequence of an identified plant gene glutamate formiminotransferase (GFT) in maize, leading to a glycine‐to‐asparagine substitution at the 228th in the protein sequence and contributing to the variation of folate accumulation in mature seeds of a maize inbred line population. This gene encodes a protein highly similar to the formiminotransferase domain of mammalian formiminotransferase cyclodeaminase. In vitro biochemical analysis of this protein reveals an activity of triggering 5‐methyl‐tetrahydrofolate (5‐M‐THF)‐to‐MeFox conversion, other than exerting an activity of formiminotransferase in mammals. Loss of ZmGFT function triples 5‐M‐THF levels, and overexpression of G‐allele‐carrying ZmGFT boosts the metabolic flow toward MeFox. Functional conservation of GFT is validated in rice and Arabidopsis. The asparagine‐to‐glycine substitution enhances 5‐M‐THF‐to‐MeFox conversion, as demonstrated by in vitro assays and in silico analyses. The functional characterization of the GFT gene has uncovered a new metabolic fate of 5‐M‐THF, apart from a C1 donor for methionine synthesis, in plants, and a distinct activity from its mammalian ortholog. The natural variation identified is useful for breeding folate‐fortified maize varieties. This work demonstrates the distinct roles of the glutamate formiminotransferase ( GFT ) enzyme in plants. The GFT triggers the conversion of 5-methyl-tetrahydrofolate to MeFox in plants, a process distinct from its role as a formiminotransferase in mammals. A key allelic variation in GFT, with the G-allele linked to high folate levels in maize, highlights its significant impact on folate metabolism and breeding. Abstract Identifying genes involved in folate accumulation is critical for elucidating the regulatory mechanisms of folate metabolism and breeding folate-rich crops. Here, a natural A-to-G variation at the 682nd bp is identified in the coding sequence of an identified plant gene glutamate formiminotransferase ( GFT ) in maize, leading to a glycine-to-asparagine substitution at the 228th in the protein sequence and contributing to the variation of folate accumulation in mature seeds of a maize inbred line population. This gene encodes a protein highly similar to the f ormimino t ransferase domain of mammalian f ormimino t ransferase c yclo d eaminase. In vitro biochemical analysis of this protein reveals an activity of triggering 5-methyl-tetrahydrofolate (5-M-THF)-to-MeFox conversion, other than exerting an activity of formiminotransferase in mammals. Loss of ZmGFT function triples 5-M-THF levels, and overexpression of G-allele-carrying ZmGFT boosts the metabolic flow toward MeFox. Functional conservation of GFT is validated in rice and Arabidopsis. The asparagine-to-glycine substitution enhances 5-M-THF-to-MeFox conversion, as demonstrated by in vitro assays and in silico analyses. The functional characterization of the GFT gene has uncovered a new metabolic fate of 5-M-THF, apart from a C1 donor for methionine synthesis, in plants, and a distinct activity from its mammalian ortholog. The natural variation identified is useful for breeding folate-fortified maize varieties. Advanced Science, Volume 12, Issue 42, November 13, 2025.