Fixing flavins: hijacking a flavin transferase for equipping flavoproteins with a covalent flavin cofactor

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing a covalent bond between a flavin cofactor and its hosting protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that ‘dissociable’ flavoproteins can be turned into covalent flavoproteins. Specifically, three different FMN-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage (LOV) domain protein, a mini singlet-oxygen-generator (miniSOG), and a nitroreductase (BtNR). Optimizing the flavinylation motif and expression conditions led to covalent flavinylation of all three flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance such as higher thermostability or catalytic performance. Crystal structures of all three covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins which cannot lose their cofactor, boosting their performance.