Developing and enhancing promiscuous activity for NAD(P)H-dependent flavin reductase via elimination of cofactor

Promiscuous enzymes have shown their synthetic abilities in generating various organic compounds with high selectively and efficiency under mild conditions. Therefore, the design and development of conditions to raise promiscuity to the enzymes have been under the spotlight in recent years. Flavin reductase, that reduces flavins by using NADH as a cofactor, has not been studied in promiscuous reactions. In the present study, it was aimed to develop a catalytic promiscuous activity in the recombinant E.coli flavin reductase by removing its cofactor. The flavin reductase demonstrated a promiscuous activity for Knoevenagel condensation and Michael addition reactions individually. The cofactor-independent promiscuous activity of the flavin reductase was further enhanced by altering the reaction conditions to proceed a Knoevenagel-Michael addition cascade for tetraketone synthesis. Yet, the presence of the cofactor blocked the promiscuous Knoevenagel condensation, Michael addition, and therefore the cascade reaction, demonstrating that the removal of NADH was pivotal in inducing the promiscuous activity. Furthermore, molecular docking and MD simulations were performed to obtain more structural and mechanistic details of the transformation. The computational studies identified the most likely catalytic sites of the flavin reductase in the reaction. Additionally, a truncated variant of the enzyme that lacked 28 residues from the C-terminus displayed comparable activity to the wild-type enzyme, indicating the robustness of the enzyme in performing the cascade reaction. In brief, the cofactor-elimination method presented in this work could be considered as a straightforward and economical approach for inducing enzyme promiscuity in promoting organic transformations.