From free to fixed: unveiling the impact of covalent FMN attachment in flavoproteins
Flavoproteins are special types of enzymes that use small helper molecules called FMN or FAD to carry out important redox reactions, the kind of chemical processes that keep many biological systems running. They play essential roles in nature and have many useful applications in biotechnology.
In this thesis, I study flavoproteins with a focus on a technique called “flavin-fixing.” This method uses a bacterial enzyme named ApbE to securely and specifically attach the FMN cofactor to proteins. By designing short recognition sequences within the protein, we can make this attachment both strong and precise. This helps enzymes hold on to their cofactors more tightly and function more efficiently.
My research explores how flavin-fixing affects enzyme activity and light-related (photochemical) properties, and how it can be applied in protein labeling, reactive oxygen species (ROS) generation, and optogenetics, the use of light to control biological processes.I also describe how this method can be used to covalently link FMN to different proteins, such as miniSOG and L-lactate oxidase/dehydrogenase, highlighting both the challenges and strategies to improve enzyme performance while maintaining stable cofactor binding.
Finally, I use mass photometry, a single-molecule technique, to show how enzymes can change shape or dissociate when they interact with ligands. This provides new insight into a possible mechanism behind the intriguing phenomenon of “enhanced enzyme diffusion.”