Site-specificity of the second-site suppressor mutation of the Asp-285-->Asn mutant of metal-tetracycline/H+ antiporter of Escherichia coli and the effects of amino acid substitutions at the first and second sites.
The deleterious effect of the mutation of Asp-285 to Asn of the metal-tetracycline/H+ antiporter (TetA) of Escherichia coli is suppressed by the second-site mutation of Ala-220 to an acidic amino acid residue (Yamaguchi, A., O'yauchi, R., Someya, Y., & Sawai, T. (1993) J. Biol. Chem. 268, 26990-26995). In this study, site-specific second-site Glu mutants as to 11 different positions around position 220 were established from the Asp-285-->Asn mutant TetA protein. Among them, only the Ala-220-->Glu mutant completely suppressed the deleterious effect of the Asp-285-->Asn mutation, indicating that position 220 is highly specific for the suppression. Although E. coli cells producing second-site Glu mutants as to positions 213, 216, 217, 218, 219, 221, 222, and 223 of the Asn-285 mutant were as tetracycline sensitive as the host cells without TetA, Gly-224-->Glu and Pro-227-->Glu second-site mutants of the Asn-285 mutant conferred low-level tetracycline resistance, the levels decreasing in this order. These two positions and position 220 are on the same side of putative transmembrane helix VII. The Phe-289-->Asp mutation, which is located at a position one-alpha-helical-turn downstream from Asp-285 in the same putative helix, IX, did not suppress the Asn-285 mutation. The introduction of an acidic residue at the second site was essential for suppression of the Asn-285 mutation because Lys-220 and Gln-220 second-site mutants of the Asn-285 mutant showed very low tetracycline resistance.(ABSTRACT TRUNCATED AT 250 WORDS)
Someya
Y
Y
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Niwa
A
A
Sawai
T
T
Yamaguchi
A
A
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001216:Asparagine; D001224:Aspartic Acid; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D018698:Glutamic Acid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic; D013752:Tetracycline / Q000378:metabolism
D017920:Antiporters; D001426:Bacterial Proteins; C079051:tetA protein, Bacteria; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D013752:Tetracycline