Riboflavin (vitamin B2) is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide, which are essential coenzymes in all free-living organisms. Riboflavin biosynthesis in many Bacteria but not in Archaea is controlled by FMN-responsive riboswitches. We identified a novel bifunctional riboflavin kinase/regulator (RbkR), which controls riboflavin biosynthesis and transport genes in major lineages of Crenarchaeota, Euryarchaeota and Thaumarchaeota. RbkR proteins are composed of the riboflavin kinase domain and a DNA-binding winged helix-turn-helix-like domain. Using comparative genomics, we predicted RbkR operator sites and reconstructed RbkR regulons in 94 archaeal genomes. While the identified RbkR operators showed significant variability between archaeal lineages, the conserved core of RbkR regulons includes riboflavin biosynthesis genes, known/predicted vitamin uptake transporters and the rbkR gene. The DNA motifs and CTP-dependent riboflavin kinase activity of two RbkR proteins were experimentally validated in vitro. The DNA binding activity of RbkR was stimulated by CTP and suppressed by FMN, a product of riboflavin kinase. The crystallographic structure of RbkR from Thermoplasma acidophilum was determined in complex with CTP and its DNA operator revealing key residues for operator and ligand recognition. Overall, this study contributes to our understanding of metabolic and regulatory networks for vitamin homeostasis in Archaea.
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