Final Report Summary - N-ABLE (Nitrogenase and Nitrous Oxide Reductase:Biomolecular Engineering of Complex Redox Enzymes)
The N-ABLE Project has addressed the reaction mechanisms and biosynthetic pathways of the complex metalloenzymes nitrous oxide reductase and nitrogenase, both of which are of significant interest for bioremediation and green biotechnology. Nitrous oxide is a critical greenhouse gas that is released in substantial quantities as a byproduct of nitrogen fertilization in modern agriculture. Ist biological conversion to inert dinitrogen gas is mediated by nitrous oxide reductase, an oxygen-sensitive copper protein. Within N-ABLE, we have investigated the biogenesis of this enzyme and have produced and characterized the individual components of the complex maturation pathway. Subsequently these modules were integrated into a functional expression system for the production of an enzymatically active, re-factored nitrous oxide reductase in E. coli, serving as a model for accordingly modified organisms for bioremediatory purposes.
The second part of N-ABLE dealt with the enzyme nitrogenase and thus addressed the problem of nitrogen fertilization from an entirely different angle. This enzyme uniquely catalyzes the biological fixation of atmospheric dinitrogen, and we were able to contribute to understanding its complex mode of action. Using the method of spatially-refined anomalous dispersion developed in the laboratory in combination with X-ray absorption spectroscopy, we were able to experimentally analyze the electronic structure of the active site cofactor and subsequently demonstrate the mode of binding of the Inhibitor CO as well as of a reaction intermediate. In parallel, the biogenesis of nitrogenases was studied with the aim to generate a recombinant, heterologous production system for the entire apparatus that might eventually serve as a basis for transferring the ability to fix N2 to Food crops, thus obliterating the requirement for nitrogen fertilizers in industrial agriculture.
The second part of N-ABLE dealt with the enzyme nitrogenase and thus addressed the problem of nitrogen fertilization from an entirely different angle. This enzyme uniquely catalyzes the biological fixation of atmospheric dinitrogen, and we were able to contribute to understanding its complex mode of action. Using the method of spatially-refined anomalous dispersion developed in the laboratory in combination with X-ray absorption spectroscopy, we were able to experimentally analyze the electronic structure of the active site cofactor and subsequently demonstrate the mode of binding of the Inhibitor CO as well as of a reaction intermediate. In parallel, the biogenesis of nitrogenases was studied with the aim to generate a recombinant, heterologous production system for the entire apparatus that might eventually serve as a basis for transferring the ability to fix N2 to Food crops, thus obliterating the requirement for nitrogen fertilizers in industrial agriculture.