Cysteine-scanning Analysis of the Nucleobase-Ascorbate Transporter Signature Motif in YgfO Permease of Escherichia coli

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).…”

“…The other two irreplaceable residues of YgfO are in the neighboring helices TM8 (Glu-272) and TM9a (Asp-304) and appear to be implicated with the conformational changes following binding and/or coupling purine with proton translocation, but not with substrate binding per se (10). Apart from the irreplaceable ones, a number of other important residues have been found, including residues at the middle of helices TM1 (His-31) and TM3 (Asn-93), which contribute to determining the proper affinity and selectivity of the purine binding site (10), residues at the middle of TM12 (Asn-430, Ile-432), which are close to the binding site and/or optimize binding indirectly (9), and residues within or adjacent to the NAT motif which are important conformationally (Ala-323) (11) or involved in defining the optimal specificity profile (Thr-332, Gly-333, Ser-336) (8,11). Overall, residues delineated as crucial for the mechanism of substrate recognition and selectivity cluster primarily at the NAT motif region (Fig.…”

“…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).…”

“…) and [8][9][10][11][12][13][14] C]uric acid (51.5 mCi mmol Ϫ1 ) were purchased from Moravek Biochemicals. Non-radioactive nucleobases and analogues were from Sigma.…”

“…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). For construction of mutants, two-stage PCR was performed on the template of Cys-less or wild-type YgfO, as indicated.…”