Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.
A carboxyl group of Asp-285 is essential for tetracycline/H+ antiport mediated by the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA) of Escherichia coli (Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M., and Sawai, T. (1992) J. Biol. Chem. 267, 7490-7498). Spontaneous tetracycline resistance revertants were isolated from E. coli cells carrying the Asn-285 mutant tetA gene. All of the revertants were due to the second-site mutation at codon 220 of GCG (Ala) to GAG (Glu). The Km value of the tetracycline transport mediated by the revertant TetA protein was about 4-fold higher than that of the wild-type, indicating that the revertant is a low affinity mutant. A Glu-220 and Asn-285 double mutant constructed by site-directed mutagenesis showed the same properties as the revertants, confirming that the mutation of Ala-220 is solely responsible for the suppression. The Asp-220 mutation of the Asn-285 mutant resulted in a lower level of restoration of the tetracycline resistance and the transport activity than in the case of the Glu-220 mutation. A single mutation replacing Ala-220 with Glu or Asp caused about a 2-4-fold decrease in the tetracycline resistance, but no crucial change in the transport activity. It is not likely that Glu-220 is required for a charge-neutralizing salt bridge because an unpaired negative charge in a Glu-220 or Asp-220 single mutant did not cause a serious change in the activity. An alternative explanation is reasonable; Asp-285 directly contributes to the binding of a cationic substrate, metal-tetracycline chelation complex, or proton, and an acidic residue at position 220 can take over the role of Asp-285.
Yamaguchi
A
A
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
O'yauchi
R
R
Someya
Y
Y
Akasaka
T
T
Sawai
T
T
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D017920:Antiporters / Q000235:genetics*; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005971:Glutamates / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000235:genetics*; D013489:Suppression, Genetic; D013752:Tetracycline / Q000378:metabolism
D000596:Amino Acids; D017920:Antiporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005971:Glutamates; D012097:Repressor Proteins; C079051:tetA protein, Bacteria; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D013752:Tetracycline; D000409:Alanine