Design and Synthesis of Artificial FAD Cofactors for the Light-Triggered Covalent Flavinylation of Flavoenzymes

Abstract

Flavoenzymes are versatile biocatalysts with extraordinary properties. They are not only vital to life by orchestrating complex transformations in biological processes but offer numerous biocatalytic opportunities. Most flavoenzymes have a noncovalently bound flavin cofactor. The cofactor is responsible for the catalytic versatility and prowess of flavoenzymes and is essential for their structure and stability. The dissociation of noncovalently bound flavin adenine dinucleotide (FAD) results in an immediate loss of activity and often an unstable apoenzyme, which reduces turnover stability and limits the broader application of flavoenzymes in the industry. Herein, we present a proof-of-concept for the light-induced covalent flavinylation of the three flavoenzymes glucose oxidase (GOx), cellobiose dehydrogenase (CDH), and cyclohexanone monooxygenase (CHMO). We designed and synthesized seven FAD analogs bearing a diazirine group, allowing the light-triggered covalent anchoring of the cofactor. One of the photoclickable FAD analogs (diazFAD4) could be integrated successfully into all flavoenzymes of interest, achieving activities comparable to those of the native FAD. The covalent bond formation was initiated by ultraviolet (UV) light irradiation and could be confirmed via fluorescence imaging. By expanding the photoaffinity labeling technique, we provide a novel method for the covalent and functional incorporation of cofactors into biocatalysts.