Role of the Charge Interaction between Arg70 and Asp120 in the Tn10-encoded Metal-Tetracycline/H+ Antiporter of Escherichia coli

Abstract

120 was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp 66 mutation. The chargeexchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg 2؉ ), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg 2؉ ; however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg

Interdomain Loop Mutation Asp190Cys of the Tetracycline Efflux Transporter TetA(B) Decreases Affinity for Substrate

“…The cysteine substitution for aspartate at position 190 showed an average K m value 3.8 times that of the wild type but did not produce any modification in V max ( Table 1). The K m and V max values obtained for the wild type are in agreement with those reported in the literature (3,8,9,14). The lower affinity for tetracycline of the mutant Asp190Cys suggests that the aspartate residue is involved in the substrate interaction.…”

Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

“…The eighth residue (alanine-323) is a conservative substitution from the serine at the analogous position in TetA(C). Furthermore, residues arginine-70 and aspartate-120 of Tet(B) were shown to be required for proper insertion of the transmembrane regions into the cytoplasmic membrane (34). These amino acids were also present at the corresponding positions in TetA (41), suggesting that these residues serve similar functions.…”

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

“…The boundaries of membrane-embedded domains have been defined (126,134), and the proposed topology of the proteins developed by earlier modeling methods has been confirmed experimentally (127). Furthermore, charge interactions between key residues such as arginine-70 and aspartate-120 in the Tet(B) protein have been identified as a requirement for correct positioning of transmembrane segments in the cytoplasmic membrane (279). Mutation studies have also been used to try to identify the antibiotic binding site within the efflux proteins from gram-negative bacteria.…”