The riboflavin kinase domain is essential for FMNAT catalysis: A linker-dependent regulatory mechanism in Staphylococcus aureus FAD synthetase

Flavin adenine dinucleotide (FAD) synthetase (FADS) from Staphylococcus aureus is a bifunctional enzyme comprising an N-terminal FMN adenylyltransferase (FMNAT) domain that catalyzes FAD synthesis and a C-terminal riboflavin kinase (RFK) domain responsible for riboflavin phosphorylation. While eukaryotic FMNAT and RFK homologs function as independently, the mechanistic basis for their fusion and interdependence in bacteria remains unclear. Here, we show that S. aureus FADS functions as an obligate structural assembly exhibiting pronounced functional asymmetry between its two domains. The isolated RFK domain retains substantial catalytic activity, with a kcat of 14.5 min-1, whereas the isolated FMNAT domain is catalytically inactive despite being properly folded. Perturbing the native inter-domain linker with flexible or rigid sequences severely compromises both domains, with FMNAT displaying a disproportionately greater functional loss. Biophysically, the isolated RFK domain exists predominantly as a dimer in solution, unlike the monomeric full-length enzyme, indicating that the native inter-domain linker and FMNAT domain influence the oligomeric state of RFK. Consistently, microscale thermophoresis shows a markedly reduced FMN affinity for the isolated FMNAT domain, and molecular dynamics simulations further indicate destabilization of the FMNAT active-site geometry without RFK. Together, these findings demonstrate that the RFK domain and native inter-domain linker act as essential structural elements stabilizing the FMNAT active site in a catalytically competent conformation, thereby enabling productive FAD synthesis and pointing to inter-domain coupling as a potentially important feature of bacterial FAD metabolism.