ROLE OF THE CONSERVED QUARTETS OF RESIDUES LOCATED IN THE N-TERMINAL 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 K(m) values of the Q54A and Q261A mutants for tetracycline were 140 and 160 muM, 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 K(m) value, whereas the Y50C mutant showed no transport activity at all. The H257Y and H257A mutants showed 30% and 8% of the wild-type activity, respectively. Surprisingly, the double mutants as to Tyr50 and His257 showed intermediate activities between those of the respective two single mutants or similar activity to one of the single mutants, suggesting that the two residues may act as a functional pair.