Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp....

Na(+)/H(+) antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na(+)/H(+) antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na(+) and Li(+) translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0-8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na(+) and Li(+), while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.