Role of intramembrane polar residues in the YgfO xanthine permease: HIS-31 and ASN-93 are crucial for affinity and specificity, and ASP-304 and GLU-272 are irreplaceable.

ABSTRACT: Using the YgfO xanthine permease of Escherichia coli as a bacterial model for the study of the evolutionarily ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family, we performed a systematic Cys-scanning and site-directed mutagenesis of 14 putatively charged (Asp, Glu, His, Lys, or Arg) and 7 highly polar (Gln or Asn) residues that are predicted to lie in transmembrane helices (TMs). Of 21 single-Cys mutants engineered in the background of a functional YgfO devoid of Cys residues (C-less), only four are inactive or have marginal activity (H31C, N93C, E272C, D304C). The 4 residues are conserved throughout the family in TM1 (His-31), TM3 (Asn-93/Ser/Thr), TM8 (Glu-272), and putative TM9a (Asp-304/Asn/Glu). Extensive site-directed mutagenesis in wild-type background showed that H31N a

SUBMITTER: Karena E 

PROVIDER: S-EPMC2782019 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature