Characterisation of the Rh50 protein from the ammonia-oxidising bacterium Nitrosomonas europaea

The transport of ammonium through the cell membranes, an essential process in all kingdoms of life, is accomplished by the ubiquitous Amt/Mep/Rh superfamily of proteins. The functional context of Amt/Mep and Rh transporters is diverse: bacteria, fungi, and plants use Amt/Mep proteins to scavenge ammonium for biosynthetic assimilation, whereas mammals use the Rh proteins for ammonium detoxification in erythrocytes, kidney, and liver tissues. While RH50 genes are widespread in eukaryotes they are present in some prokaryotes: an example is a chemolithoautotroph Nitrosomonas europaeawhich gains all its energy from the oxidation of ammonia to nitrate.

While Amt/Mep/Rh proteins have divergent physiological functions, they are structurally very similar, which raises the important question about the universality of the transport mechanism. We have recently proposed an elegant new model for the mechanism of electrogenic ammonium transport in bacteria Amt protein: after deprotonation of NH4+ at the periplasmic side of the transporter, a previously undiscovered polar conduction route enables H+ transfer into the cytoplasm. A parallel pathway, lined by hydrophobic groups within the protein core, facilitates the simultaneous transfer of uncharged NH3. In this context, we propose to elucidate at the molecular level the mechanism of ammonium translocation through rhesus protein from Nitrosomonas europaeaand establish whether there is a universal mechanism for biological ammonium transport. Beyond the elucidation of a central biological process, this work has important medical implications, as some Rh mutations have been associated with human pathologies. We propose to demonstrate how specific Rh mutations affect the activity of the protein to establish the relationship between Rh malfunction and the associated diseases.