His257 Is a Uniquely Important Histidine Residue for Tetracycline/H+ Antiport Function but Not Mandatory for Full Activity of the Transposon Tn10-Encoded Metal−Tetracycline/H+ Antiporter

Abstract

His257 is the only histidine residue located in the putative transmembrane region of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA) and contributes to the substrate/H+ coupling [Yamaguchi, A.. Adachi, K., Akasaka. T., Ono. N., & Sawai, T. (1991) J. Biol. Chem. 266, 6045-6051]. Tn10-TetA contains five histidine residues, including His257. When these histidine residues were replaced by alanine one by one, only the His257Ala mutant showed almost complete loss of the tetracycline transport activity, whereas the other four His --> Ala mutants, H42A, H158A, H329A, and H359A, retained transport activity comparable to that of the wild type. The mutant which contains only one histidine, His257, retained about 80% of the wild-type activity, whereas the histidine-less mutant, in which all five histidine residues were replaced by Ala, exhibited little activity. These results clearly indicated that His257 is a unique histidine residue in TetA responsible for the transport activity. The His257Tyr mutant, irrespective of the presence or absence of the other four histidine residues, retained about 30% of the wild-type tetracycline transport activity and showed corresponding tetracycline-coupled H+ translocation, indicating that an imidazole group is not necessary at position 257 for the substrate/H+ coupling. A histidine-specific reagent, diethyl pyrocarbonate (DEPC), equally inactivated the wild-type and one-histidine mutant TetA, whereas the H257Y mutant was hardly inactivated by DEPC irrespective of the presence or absence of the other four histidine residues, indicating that the inactivation by DEPC is due to the modification of His257.