Abstract
NanT and NanX are bacterial transporters that import the sialic acids, N ‐acetylneuraminate and 2,7‐anhydro‐ n ‐acetylneuraminate, respectively. Here, we used complementary biophysical and computational approaches to structurally characterise Escherichia coli NanX. Size exclusion chromatography, analytical ultracentrifugation and low‐resolution cryo‐electron microscopy reveal that NanX exists in both monomeric and dimeric states following purification. Molecular modelling and substrate docking identify key residues likely involved in 2,7‐anhydro‐ n ‐acetylneuraminate recognition. Using this information, we engineered a mutant NanX transporter that can import the NanT‐specific substrate, N ‐acetylneuraminate, which we verified using a bacterial growth assay. These data identify amino acids involved in major facilitator superfamily mediated sialic acid transport and offer a new research perspective of its metabolism.