The role of copper proteins in the ammonia oxidation pathway of Nitrosopumilus maritimus

The contribution of ammonia oxidising archaea (AOA) to the global nitrogen cycle has become apparent in recent years. Where previously thought to be driven by bacteria, archaea may outcompete bacteria, particularly in low nutrient environments like unfertilised soils. Ammonia oxidising bacteria (AOB) are well characterised, with ammonia oxidising pathways relying predominantly on proteins utilising haem c active sites. However, homologues for these proteins (or indeed genes for any proteins containing the characteristic haem c CXXCH motif) are absent in AOA, suggesting a completely different pathway in AOA. Novel Cu-containing multi copper oxidase homologue proteins in Nitrosopumilus maritimus, have been suggested as candidates for ammonia oxidation in AOA. Bioinformatic analysis of three of these proteins lead to the prediction that; Nmar1131 is a homotrimeric laccase-like 2dMCO, Nmar1354 a homotrimeric 3dMCO with an additional T1Cu site in the C-terminus cupredoxin domain, and Nmar1667 an NO-forming nitrite reductase. Purification of Nmar1131 was performed. Originally over-expression was attempted in a cytoplasmic vector, which proved unsuccessful. To echo the predicted native periplasmic nature of this protein, the nmar1131 gene was cloned into pET26b, a vector containing a periplasmic signal peptide which yielded purified protein. Data from this study, could provide important insights into how to mitigate anthropogenic nitrogen inputs into the environment such as from modern agricultural practices.