Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport.
The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.
Yamaguchi
A
A
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Ono
N
N
Akasaka
T
T
Noumi
T
T
Sawai
T
T
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004151:Dipeptides / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D013752:Tetracycline / Q000378:metabolism*
D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004151:Dipeptides; D015345:Oligonucleotide Probes; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D001224:Aspartic Acid; D012694:Serine; D013752:Tetracycline