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

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Transport

Background:

• The Tn10-encoded metal-tetracycline/H+ antiporter (TetA) facilitates tetracycline transport across membranes.
• TetA contains five histidine residues, with His257 located in a transmembrane region.

Purpose of the Study:

• To investigate the specific role of His257 in TetA-mediated tetracycline transport.
• To determine if the imidazole group of His257 is essential for substrate/H+ coupling.

Main Methods:

• Site-directed mutagenesis of TetA to replace histidine residues with alanine or tyrosine.
• Assessing tetracycline transport activity and tetracycline-coupled H+ translocation in wild-type and mutant TetA proteins.
• Inactivation of TetA activity using the histidine-specific reagent diethyl pyrocarbonate (DEPC).

Main Results:

• Mutagenesis of His257 to alanine resulted in a near-complete loss of tetracycline transport activity.
• Mutations at other histidine residues (H42A, H158A, H329A, H359A) did not significantly affect transport activity.
• The His257Tyr mutant retained partial transport activity, indicating the imidazole group is not strictly required for coupling.
• DEPC inactivated wild-type and single-histidine TetA but not the H257Y mutant, confirming His257 modification as the cause of inactivation.

Conclusions:

• His257 is uniquely essential for TetA's tetracycline transport activity.
• The imidazole group of His257 is not strictly necessary for substrate/H+ coupling.
• His257 is the primary target for DEPC-induced inactivation of TetA.