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 intrahelical 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 32Pi by homologous Pi/Pi 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 Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-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.
Hall
J A
JA
Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21205, USA.
Fann
M C
MC
Maloney
P C
PC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D013379:Substrate Specificity / Q000235:genetics; D013403:Sugar Phosphates / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D013403:Sugar Phosphates; C063498:UhpT protein, E coli