Origin and Evolution of Key Enzymes in the Anammox Pathway Revisited

Anaerobic ammonium oxidizing bacteria in the class "Candidatus Brocadiia" in the Planctomycetota are the only known group of bacteria capable of producing energy by coupling the oxidation of ammonium to the reduction of nitrite within a unique bacterial organelle called the anammoxosome. Due to the lack of homologs in other species, it is hypothesized that the key enzyme in this process, the hydrazine synthase complex, originated by de novo birth. We performed extensive searches for proteins that exhibited similarity in sequence and structure to the hydrazine synthase subunits and identified distantly related homologs in anaerobic bacteria from the phyla Planctomycetota and Desulfobacterota. However, key residues of importance for the enzymatic function were not conserved, rejecting the hypothesis that the identified genes represent previously unrecognized anammox bacteria. Phylogenetic analyses indicate that the anammox pathway has been assembled from genes acquired by horizontal gene transfer from a variety of anaerobic bacteria. The ancestral states of enzymes in the hydroxylamine oxidoreductase family were inferred, and transitions between reductive and oxidative forms of the enzymes were mapped onto the phylogenetic tree. Finally, it is shown that the signal sequences of key enzymes in the anammox pathway are able to transport a reporter gene into the periplasm of Escherichia coli cells. In conclusion, our findings suggest that the hydrazine synthase complex has evolved from already existing heme-binding periplasmic proteins and that the anammoxosome has an endogenous origin.