Identification of Two Essential Arginine Residues in UhpT, the Sugar Phosphate Antiporter of Escherichia coli

Abstract

Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (>/=30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity.

Analysis of Substrate-Binding Elements in OxlT, the Oxalate:Formate Antiporter of Oxalobacter formigenes

“…In each case we confirmed the discrepancies noted in the preliminary screen. Thus, for malonate we obtained K iapp values (mean ± SE) of 6.1 ± 0.4 mM for the parental protein, comparable to that found earlier (25), but significantly elevated values of 115 ± 3.6 mM and 51 ± 13 mM for the modified R272C and K355C proteins, respectively (Fig. 4A).…”

“…This conclusion is reinforced by findings related to OxlT stability, which show that MTSEA modification restores the capacity for substrate (oxalate) stabilization (Fig. 2), a phenomenon that requires ligand binding (18,25).…”

“…Recent study of the MFS nitrate transporter, NtrA, suggested that paired arginine residues, one each in TM2 and TM8, might serve as substrate (anion) binding sites (26) as in GlpT and UhpT (14,25), and this raised the possibility that OxlT and its variants might accept inorganic as well as organic anions. In 10 added tests, therefore, we examined a panel of inorganic anions as possible OxlT substrates, including bicarbonate, chloride, dithionite, fluoride, nitrate, nitrite, perchlorate, perchlorite, phosphate, sulfate, sulfite, and thiocyanate.…”

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

“…In particular, our work confirms the presence in OxlT of a bifunctional active site, comprised of two positively charged residues (Arg-272 and Lys-355), positioned 10 Å apart across the permeation pathway. This arrangement bears striking resemblance to that found in GlpT, where the anion-binding site incorporates two arginine residues separated by a comparable distance spanning the pathway (9,17). Indeed, superposition of the GlpT and OxlT ligand-binding residues shows these active site components all point to the same region, together identifying a discoid roughly 10 Å in diameter and 3 Å in thickness as the main region involved in substrate (anion) binding.…”

“…The center of the GlpT substrate permeation pathway contains two basic residues (Arg-46, Arg-269) that serve as ligand-binding sites for inorganic phosphate or glycerol 3-phosphate (9,17). The OxlT homology model suggests a parallel finding for OxlT, which requires both Arg-272 (Fig.…”