Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices.
Three conserved aspartyl residues located in the putative transmembrane helices in the Tn10-encoded metal-tetracycline/H+ antiporter were replaced by Asn, Lys, or Glu with oligonucleotide-directed site-specific mutagenesis. Replacement of Asp84 or Asp15 by Asn or Lys caused a severe defect in tetracycline transport activity, however, the Glu84 and Glu15 mutants retained 150 and 40% of the wild type activity, respectively, indicating the critical role of the negative charge. The increase in the activity of the Glu84 mutant was due to an increase in the affinity for the substrate. H+/tetracycline coupling was intact in these mutants, including Asn and Lys mutants. On the other hand, all of the Asp285-substitution mutants showed a severe defect in tetracycline transport activity and a complete lack of tetracycline-coupled H+ transport. However, since in vivo tests showed the tetracycline resistance for the Glu285 mutant, a negative charge in position 285 plays some role in maintaining the possible down-hill and/or low affinity efflux of accumulated tetracycline from intact cells. Similar work was done for Asp365, and here the Asn and Glu mutants showed decreased but high activity, while the Lys mutant was only marginally active (5%), indicating that a negative charge is not so demanding in position 365, possibly because it is not in the membrane.
Yamaguchi
A
A
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Akasaka
T
T
Ono
N
N
Someya
Y
Y
Nakatani
M
M
Sawai
T
T
D000595:Amino Acid Sequence; D017920:Antiporters; D001224:Aspartic Acid / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001693:Biological Transport, Active; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D011522:Protons; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000378:metabolism*; D013753:Tetracycline Resistance / Q000235:genetics
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D008670:Metals; D011522:Protons; D012097:Repressor Proteins; C079051:tetA protein, Bacteria; D001224:Aspartic Acid; D013752:Tetracycline