Cloning and functional characterization of two bacterial members of the NAT/NCS2 family inEscherichia coli

Abstract

The coding potential of the genome of E. coli K-12 includes YgfO and YicE, two members of the evolutionarily conserved NAT/NCS2 transporter family that are highly homologous to each other (45% residue identity) and closely related to UapA of Aspergillus nidulans, a most extensively studied microbial member of this family. YgfO and yicE were cloned from the genome, over-expressed extrachromosomally and assayed for uptake of [(3)H]xanthine and other nucleobases, in E. coli K-12, under conditions of negligible activity of the corresponding endogenous systems. Alternative, essentially equivalent functional versions of YgfO and YicE were engineered by C-terminal tagging with an epitope from the E. coli lactose permease and a biotin-acceptor domain from Klebsiella pneumoniae. Both YgfO and YicE were shown to be present in the plasma membrane of E. coli and function as specific, high-affinity transporters for xanthine (K(m) 4.2-4.6 microM for YgfO, or 2.9-3.8 microM for YicE), in a proton motive force-dependent manner; they display no detectable transport of uracil, hypoxanthine, or uric acid at external concentrations of up to 0.1 mM. Both YgfO and YicE are inefficient in recognizing uric acid or xanthine analogues modified at position 8 of the purine ring (8-methylxanthine, 8-azaxanthine, oxypurinol, allopurinol), which distinguishes them from their fungal homologues UapA and Xut1.

Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

“…More than 1,000 sequence entries are known but few are functionally characterized to date. Structure-function relationships have been studied extensively in two members, the eukaryotic UapA, a high-affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (3)(4)(5)(6) and the prokaryotic YgfO, a specific, high-affinity xanthine:H ϩ symporter from Escherichia coli (7)(8)(9)(10)(11). Mutagenesis data from both lines of study have shown that key NAT determinants are strikingly similar between the two transporters, and that few residues conserved throughout the family may be invariably critical for function (10).…”

“…Topology model of YgfO permease. The model is based on program TMHMM, evidence that C terminus is cytoplasmic (7,15) and our unpublished evidence (see footnote 3) on the accessibility of loops to hydrophilic reagents. Irreplaceable residues of YgfO (10) are numbered and bolded.…”

“…For kinetic uptake measurements, initial rates were assayed in T184 cells, at 5-20 s, in the concentration range of 0.1-100 M [ For assaying the effect of NEM on xanthine uptake activity, T184 cells were preincubated with NEM at the indicated conditions, excess reagents and ligands were removed by centrifugation, and transport assays were performed in the presence of phenazine methosulfate (PMS) (0.2 mM) and potassium ascorbate (20 mM) (8). For ligand competition experiments, uptake of [ 3 H]xanthine (1 M) was assayed in the absence or presence of unlabeled analogues (1 mM) (7). For kinetic analysis, putative inhibitors were used in the concentration range of 1 M to 2 mM, and data were fitted to the equation y ϭ B ϩ (T Ϫ B)/(1 ϩ 10 ((log IC50 Ϫ log x) h) ) for sigmoidal dose-response (variable slope), where x is the concentration variable, y (activity) values range from T (top) to B (bottom) and h is the Hill coefficient, for sigmoidal dose-response (variable slope), using Prism4, to obtain IC 50 values; in all cases, the Hill coefficient was close to Ϫ1, consistent with presence of one binding site.…”

“…DNA Manipulations-Construction of expression plasmids and BAD (biotin-acceptor domain)-tagged versions of YgfO has been described (7). For construction of Cys-less YgfO, the five native Cys codons were replaced simultaneously with Ser codons, using two-stage (multiple overlap/extension) PCR on the template of wild-type YgfO tagged at C terminus with the BAD tag (8).…”

“…Transport Assays and Kinetic Analysis-E. coli T184 were assayed for active transport of [ 3 H]xanthine (1 M), by rapid filtration, at 25°C, pH 7.5, as described (7). For kinetic uptake measurements, initial rates were assayed in T184 cells, at 5-20 s, in the concentration range of 0.1-100 M [ For assaying the effect of NEM on xanthine uptake activity, T184 cells were preincubated with NEM at the indicated conditions, excess reagents and ligands were removed by centrifugation, and transport assays were performed in the presence of phenazine methosulfate (PMS) (0.2 mM) and potassium ascorbate (20 mM) (8).…”