Alteration of Na+-coupled transport in site-directed mutants of the melibiose carrier of Escherichia coli

摘要

Asn-58 of the Escherichia coli melibiose carrier was replaced by Ala, Leu, Ser, and Gln. Trp-54 was replaced by Leu and a double mutant Leu-54/Ala-58 was constructed using site-directed mutagenesis. Cation/sugar cotransport and sugar-induced cation uptake were studied for each mutant. The charge of Asn-58 to Ala results in a nearly complete loss of Na+-stimulated galactoside transport as well as sugar-stimulated Na+ uptake. Substitutions of Leu, Gln, and Ser for Asn-58 were also defective in Na+-stimulated sugar transport. The Trp-54 to Leu mutant shows moderate sugar accumulation with cation selectivity similar to wild-type. The double mutant Leu-54/Ala-58 shows elevated H+-melibiose cotransport as well as reduced Na+-stimulated melibiose cotransport. These results suggest that Asn-58 is important for Na+ recognition.