Flavin Fixing in Old Yellow Enzyme from Thermus scotoductus: A Comparative Study of the Wild-Type Enzyme and Covalently Flavinylated Mutants

Ene reductases, belonging to the Old Yellow Enzyme (OYE) family, are widely used for biocatalysis. The OYE from Thermus scotoductus SA-01 (TsOYE) gained great attention due to its broad substrate scope, high stereoselectivity, thermostability, and catalytic versatility. Recently, the otherwise noncovalently bound flavin cofactor (FMN) was covalently anchored in several TsOYE mutants using the “flavin-fixing” method. However, the biochemical properties of these mutants remained unexplored. A detailed comparative study of wild-type (WT) TsOYE and the flavin-fixing variant F1 (F1 TsOYE) revealed that F1 TsOYE has a lower stability and poorer catalytic activity. Interestingly, both WT and F1 TsOYE have comparable redox potential values. These results suggest that the decrease in activity and stability is primarily caused by changes in structure and structural dynamics induced by the mutations and the covalent flavin-protein linkage. Replacing residues in the flavinylation recognition site did not result in significant repair of enzyme activity. Our findings highlight the sensitivity of TsOYE activity to covalent FMN incorporation and its associated mutations and underscore the necessity of structural insights for further rational design. This study also provides critical groundwork for optimizing the flavin-fixing strategy.