It has been well established that VaNhaB, a NhaB-type Na(+)/H(+) antiporter found in Vibrio alginolyticus, exhibits a striking acid sensitivity. However, the molecular basis of the pH-dependent regulatory mechanism of the antiport activity is yet to be investigated. In this study, we generated various chimeric proteins composed of VaNhaB and a pH insensitive ortholog found in Escherichia coli (EcNhaB) and analyzed the pH responses of their Na(+)/H(+) antiport activities to search for the key residues or domains that are involved in the pH sensitivity of VaNhaB. Our results revealed the significant importance of a stretch of amino acid residues within the loop 8-loop 9 regions (E373-D434) responsible for the acid sensitivity of VaNhaB, along with the possible involvement of other unidentified residues that are widely spread in the primary structure of VaNhaB. Moreover, we demonstrated that the E373-D434 region of VaNhaB was able to confer some degree of acid sensitivity on our pH insensitive chimeric antiporter that is mainly composed of EcNhaB except for seven amino acid substitutions at the N-terminal end. This result strongly suggested the possibility that the E373-D434 region is able to act, at least partially, as machinery that diminishes the activity of the NhaB-type antiporter at an acidic pH.

NhaB-like antiporters were the second described class of Na + /H + antiporters, identified in bacteria more than 20 years ago. While nhaB-like gene sequences have been found in a number of bacterial genomes, only a few of the NhaB-like antiporters have been functionally characterized to date. Although earlier studies have identified a few pH-sensitive and -insensitive NhaB-like antiporters, the mechanisms that determine their pH responses still remain elusive. In this study, we sought to investigate the diversities and similarities among bacterial NhaB-like antiporters, with particular emphasis on their pH responsiveness. Our phylogenetic analysis of NhaB-like antiporters, combined with pH profile analyses of activities for representative members of several phylogenetic groups, demonstrated that NhaB-like antiporters could be classified into three distinct types according to the degree of their pH dependencies. Interestingly, pH-insensitive NhaB-like antiporters were only found in a limited proportion of enterobacterial species, which constitute a subcluster that appears to have diverged relatively recently among enterobacterial NhaB-like antiporters. Furthermore, kinetic property analyses of NhaB-like antiporters at different pH values revealed that the degree of pH sensitivity of antiport activities was strongly correlated with the magnitude of pH-dependent change in apparent K m values, suggesting that the dramatic pH sensitivities observed for several NhaB-like antiporters might be mainly due to the significant increases of apparent K m at lower pH. These results strongly suggested the possibility that the loss of pH sensitivity of NhaB-like antiporters had occurred relatively recently, probably via accumulation of the mutations that impair pH-dependent change of K m in the course of molecular evolution.

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