Role of the conserved quartets of residues located in the N- and C-terminal halves of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli.
In the putative secondary structure of the transposon Tn10-encoded metal-tetracycline/H+ antiporter [Yamaguchi, et al. (1992) J. Biol. Chem. 267, 7490-7498], Tyr50-XXX-Gln54 in transmembrane helix 2 and Gly80-XXX-Asp84 in helix 3 are thought to face each other in the N-terminal region. At the corresponding positions in the C-terminal region, a similar quartet of residues, His257-XXX-Gln261 in helix 8 and Gly281-XXX-Asp285 in helix 9, is also located. The quartets involve the residues Asp84 and Asp285, which have been revealed to be essential for the tetracycline transport function. When Gln54 and Gln261 were replaced with Ala by site-directed mutagenesis, the active tetracycline transport activity decreased to about 10% and 40% of the wild-type level, respectively. The Km values of the Q54A and Q261A mutants for tetracycline were 140 and 160 microM, respectively, which are about 8-fold higher than that of the wild type. Thus, the two Gln residues may contribute to the substrate recognition. On the other hand, the replacement of Gly80 and Gly281 with Leu caused a complete loss of the transport activity, whereas the G80A and G281A mutants retained about 10% and 30% of the activity of the wild-type, respectively, suggesting that a bulky side chain at positions 80 and 281 causes steric hindrance of the transport. The mutation of Tyr50 to Ala or His caused a decrease in activity by a factor of about 3 without a significant change in the Km value, whereas the Y50C mutant showed no transport activity at all.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
A
A
Yamaguchi
T
T
Akasaka
T
T
Kimura
T
T
Sakai
Y
Y
Adachi
T
T
Sawai
D000595:Amino Acid Sequence; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane; D003062:Codon / Q000235:genetics; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017433:Protein Structure, Secondary; D011815:R Factors; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013752:Tetracycline / Q000378:metabolism
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003062:Codon; D004251:DNA Transposable Elements; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; C079051:tetA protein, Bacteria; D013752:Tetracycline