Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor

Area of Science:

• Biochemistry
• Protein Engineering
• Enzymology

Background:

• Flavin-dependent enzymes typically utilize dissociable flavin cofactors.
• Cofactor dissociation can limit enzyme stability and performance.
• Developing methods for stable cofactor attachment is crucial for biotechnological applications.

Purpose of the Study:

• To engineer flavoproteins with a covalent bond between the flavin cofactor and the host protein.
• To demonstrate the feasibility of creating stable, covalent flavoproteins from initially dissociable ones.
• To assess the impact of covalent flavinylation on protein function, stability, and performance.

Main Methods:

• Engineered flavinylation motifs into target flavoproteins.
• Utilized a flavin transferase for in vivo covalent flavinylation.
• Expressed and purified engineered proteins.
• Characterized covalent flavoproteins using structural (crystallography) and functional assays.

Main Results:

• Successfully achieved covalent flavinylation of four diverse flavoproteins (LOV, mini-SO, NRE, OYE).
• Optimized flavinylation motifs and expression conditions for efficient covalent linkage.
• Engineered covalent flavoproteins maintained or improved function, thermostability, and catalytic performance.
• Crystal structures confirmed the covalent threonyl-phosphate linkage.

Conclusions:

• A novel and versatile method for creating covalent flavoproteins was established.
• This approach overcomes cofactor dissociation issues, enhancing protein robustness.
• The engineered covalent flavoproteins exhibit improved stability and performance, broadening their potential applications.