Phosphatase from the Haloacid Dehalogenase (HAD) Superfamily Catalyze the Elusive Dephosphorylation Step of Riboflavin Biosynthesis in Arabidopsis thaliana Chloroplasts

Flavin nucleotides (FMN and FAD) are essential cofactors that are involved in numerous redox reactions and other biological processes including phototropism, photorepair of DNA and circadian clocks. Riboflavin (vitamin B2) is the direct precursor of flavin nucleotides and its biosynthesis in plants has not been fully elucidated. The long-term goal of our research is to advance fundamental understanding of plant flavin metabolism, and to enable crop enrichment in vitamin B2. In plants, enzymes catalyzing all but one step on the riboflavin biosynthesis pathway have been identified. The dephosphorylation step has been a long-standing gap on this pathway, because the enzymes hydrolyzing 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5’-phosphate (ARPP) are not well understood. The goal of this work is to identify and characterize the enzyme that catalyzes the dephosphorylation reaction on riboflavin biosynthesis pathway, from the model plant Arabidopsis thaliana. Towards that goal, we cloned and purified eight candidate ARPP phosphatases belonging to Haloacid Dehalogenase (HAD) Superfamily. In vitro screening found two ARPP phosphatases (AtcpFHy/PyrP1 and AtPyrP2) catalyze the dephosphorylation reaction. Plastidial subcellular localization of AtcpFHy/PyrP1 and AtPyrP2 were confirmed and their biochemical features were studied. The physiological roles of AtcpFHy/PyrP1 and AtPyrP2 on flavin metabolism and plants growth were studied by modifying the target gene expression. T-DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas suppression of AtPyrP2 reduces flavin content and affects plant growth.