Regulating Transport Preferences of Fucosylated Sugars: Revealing Transport Mechanisms via Sugar Efflux Transporter A Transformation Into a “Tight‐in, Tight‐out” Mode

Sugar transporters play a crucial role in cellular metabolism across diverse organisms, regulating essential biological processes through efficient substrate transport. Despite extensive research efforts, the structures and mechanisms of transporters responsible for sugars have remained elusive. In this study, we investigated the transport efficiency of the Escherichia coli sugar efflux transporter A (SetA) for lactose and fucosylated lactose. By employing site and combinatorial mutations, we obtained a mutant exhibiting approximately sixfold enhanced transporter efficiency for fucosylated lactose while retaining its potency for lactose transport. In this mutant, the fundamental amino acids responsible for recognizing the galactosyl moiety remained unchanged, yet the introduction of two face‐to‐face aromatic ring residues facilitated the enhanced recognition of the fucosyl moiety. This indicated the transformation of SetA from a universal transporter into a specific “tight‐in, tight‐out” transporter. Utilizing SetA‐based structural modeling, we mapped and investigated mutations associated with diseases. The structural and biochemical insights from SET in this study offer a valuable investigating framework for understanding substrate specificity mechanisms of fucosylated sugar transporters and, by extension, other transporters in broader contexts.