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Analysis of conserved NCS 2 motifs in the E scherichia coli xanthine permease XanQ
Author(s) -
Karena Ekaterini,
Tatsaki Ekaterini,
Lambrinidis George,
Mikros Emmanuel,
Frillingos Stathis
Publication year - 2015
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.13138
Subject(s) - biology , xanthine , permease , biochemistry , transmembrane domain , conserved sequence , transporter , homology modeling , binding site , mutagenesis , stereochemistry , amino acid , peptide sequence , gene , mutation , chemistry , enzyme
Summary The xanthine permease XanQ of E scherichia coli is a paradigm for transporters of the evolutionarily broad family nucleobase‐cation symporter‐2 ( NCS 2) that transport key metabolites or anti‐metabolite analogs. Most functionally known members are xanthine/uric acid transporters related to XanQ and belong to a distinct phylogenetic cluster of the family. Here, we present a comprehensive mutagenesis of XanQ based on the identification and C ys‐scanning analysis of conserved sequence motifs in this cluster. Results are interpreted in relation to homology modeling on the structurally known template of UraA and previous data on critical binding‐site residues in transmembrane segments ( TMs ) 3, 8 and 10. The current analysis, of motifs distant to the binding site, revealed a set of functionally important residues in TMs 2, 5, 12 and 13, including seven irreplaceable ones, of which six are G ly residues in the gate domain (159, 369, 370, 383, 409) and in TM 2 ( G ly‐71), and one is polar ( G ln‐75). G ln‐75 ( TM 2) is probably crucial in a network of hydrogen‐bonding interactions in the middle of the core domain involving another essential residue, A sp‐304 ( TM 9). Although the two residues are irreplaceable individually, combinatorial replacement of G ln‐75 with Asn and of A sp‐304 with Glu rescues significant transport activity.