Design and Synthesis\nof Artificial FAD Cofactors for\nthe Light-Triggered Covalent Flavinylation of Flavoenzymes

Flavoenzymes are\nversatile biocatalysts with extraordinary\nproperties.\nThey are not only vital to life by orchestrating complex transformations\nin biological processes but offer numerous biocatalytic opportunities.\nMost flavoenzymes have a noncovalently bound flavin cofactor. The\ncofactor is responsible for the catalytic versatility and prowess\nof flavoenzymes and is essential for their structure and stability.\nThe dissociation of noncovalently bound flavin adenine dinucleotide\n(FAD) results in an immediate loss of activity and often an unstable\napoenzyme, which reduces turnover stability and limits the broader\napplication of flavoenzymes in the industry. Herein, we present a\nproof-of-concept for the light-induced covalent flavinylation of the\nthree flavoenzymes glucose oxidase (GOx), cellobiose dehydrogenase\n(CDH), and cyclohexanone monooxygenase (CHMO). We designed and synthesized\nseven FAD analogs bearing a diazirine group, allowing the light-triggered\ncovalent anchoring of the cofactor. One of the photoclickable FAD\nanalogs (<b>diazFAD4</b>) could be integrated successfully into\nall flavoenzymes of interest, achieving activities comparable to those\nof the native FAD. The covalent bond formation was initiated by ultraviolet\n(UV) light irradiation and could be confirmed via fluorescence imaging.\nBy expanding the photoaffinity labeling technique, we provide a novel\nmethod for the covalent and functional incorporation of cofactors\ninto biocatalysts.