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.
Highlights
YgfU is homologous to nucleobase transporters of the ubiquitous family nucleobase-cation symporter-2 (NCS2)
YgfU Is a Uric Acid Transporter—The nucleobase-ascorbate transporter (NAT) genes ygfU, rutG, ybbY, ygfQ, yjcD, yicO, and purP were mobilized from the E. coli K-12 genome, transferred to transcriptional control of the lacZ promoter/operator in plasmid vector pT7-5, and induced for overexpression in E. coli T184 at conditions of negligible endogenous activity of oxidized purine or uracil uptake [4]
Our data show that with one exception, all NAT constructs can be expressed in the E. coli membrane at high levels, comparable with the ones of XanQ used as a control (Fig. 1B), whereas transport activity at 25 °C is observed only with YgfU, which transports [14C]uric acid (1 mM) to high levels, and RutG, which has a marginally detectable activity for [3H]xanthine (1 M) (Fig. 1C)
Summary
YgfU is homologous to nucleobase transporters of the ubiquitous family NCS2. Results: YgfU transports uric acid. We subjected YgfU to site-directed mutagenesis, based on data available for the homologous xanthine permease XanQ, and found that residues irreplaceable for the mechanism occur at five highly conserved positions, whereas a single-amino acid replacement (T100A) converts the uric acid-selective YgfU to a dual-selectivity transporter for both uric acid and xanthine.
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