Cysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helix.

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys.