IDENTIFICATION OF 2 ESSENTIAL ARGININE RESIDUES IN UHPT, THE SUGAR-PHOSPHATE ANTIPORTER OF ESCHERICHIA-COLI

Three lines of evidence indicate that arginine-46 (R46) and arginine-2 75 (R275) are essential to the function of UhpT, the Pi-linked antipor t protein of Escherichia coli. A role for arginine was initially sugge sted by the sensitivity of UhpT to inhibition by 2,3-butanedione, an a rginine-directed probe. Since the presence of substrate protected agai nst 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 gener ate a cysteine or a lysine derivative. With two exceptions (R46, R275) , all arginines could be replaced by either cysteine (10 of 14 residue s) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by exam ining a multiple alignment of the eleven known UhpT-related proteins ( greater than or equal to 30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this g roup of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine res idues (R46, R275) whose presence is essential to function. Prior bioch emical work had placed R275 at the external entrance to the translocat ion pathway, and a symmetry argument emerging from the multiple alignm ent suggests a similar position for R46. Accordingly, by virtue of the ir locations at the entrance to this pathway, we speculate that R46 an d R275 function in establishing substrate specificity.