Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.
Bottom Line: Of the 38 conserved positions tested, 24 had small effects on metal tolerance.Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function.In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).
Affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.Show MeSH
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Mentions: To investigate the role of the two glutamates in Cu+/Ag+ ATPases, mutants were generated in three such pumps: Rhizobium radiobacter CopA and CopB and Pyrococcus furiosus CopA (Lewinson et al., 2009). In all three proteins, Cu+-tolerance was not greatly affected by mutations in either of the glutamates (Table 2). In contrast, Ag+-tolerance was reduced by the mutations (Fig. 6, Table 2). The most pronounced effects were of the E270A mutation in pfCopA and of E297A of rrCopB. In these two cases the growth of the mutant was very similar to the growth of cells transformed with an empty (control) plasmid (Fig. 6, Table 2). A complete failure to grow was observed with an rrZntA mutant where both glutamates were mutated to alanines. The growth of this mutant was indistinguishable from that of the negative control, with both Zn2+ and Cd2+ (Table 1).
Affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.