Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp³⁸⁸ or Lys³⁹¹ but not if both are replaced. This suggests that Asp³⁸⁸ and Lys³⁹¹ are involved in an intra- helical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys³⁹¹ (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of ³²P(i) by homologous P(i)/P(i) exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the P(i)/P(i) exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, P(i)/P(i) exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related P(i)-linked antiporter, which lacks the Asp³⁸⁸-Lys³⁹¹ salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.