title: Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli.
abstract: The ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family includes more than 2,000 members, but only 15 have been characterized experimentally. Escherichia coli has 10 members, of which the uracil permease UraA and the xanthine permeases XanQ and XanP are functionally known. Of the remaining members, YgfU is closely related in sequence and genomic locus with XanQ. We analyzed YgfU and showed that it is a proton-gradient dependent, low-affinity (K(m) 0.5 mM), and high-capacity transporter for uric acid. It also shows a low capacity for transport of xanthine at 37 °C but not at 25 °C. Based on the set of positions delineated as important from our previous Cys-scanning analysis of permease XanQ, we subjected YgfU to rationally designed site-directed mutagenesis. The results show that the conserved His-37 (TM1), Glu-270 (TM8), Asp-298 (TM9), and Gln-318 and Asn-319 (TM10) are functionally irreplaceable, and Thr-100 (TM3) is essential for the uric acid selectivity because its replacement with Ala allows efficient uptake of xanthine. The key role of these residues is corroborated by the conservation pattern and homology modeling on the recently described x-ray structure of permease UraA. In addition, site-specific replacements at TM8 (S271A, M274D, V282S) impair expression in the membrane, and V320N (TM10) inactivates the permease, whereas R327G (TM10) or S426N (TM14) reduces the affinity for uric acid (4-fold increased K(m)). Our study shows that comprehensive analysis of structure-function relationships in a newly characterized transporter can be accomplished with relatively few site-directed replacements, based on the knowledge available from Cys-scanning mutagenesis of a prototypic homolog.
authors.affiliation: Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Papakostas
authors.forename: Stathis
authors.initials: S
authors.lastname: Frillingos
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D033702:Nucleobase Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014527:Uric Acid / Q000378:metabolism*; D019820:Xanthine / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D033702:Nucleobase Transport Proteins; C573934:YgfU protein, E coli; D019820:Xanthine; D014527:Uric Acid