The manufacture of nitramine-containing compounds, including several high-energy compounds used for military-grade explosives and propellants, can produce hazardous waste streams. Identifying enzymes to make these compounds would enable the cleaner production of them. There are no known nitramine biosynthesis pathways to date, however, some bacteria make a nitramine-containing compound called Nnitroglycine (NNG). The objective of this project is to identify all of the component enzymes of the NNG biosynthetic pathway.

This project will differentiate, by means of in vitro reconstitution, between several pathways that have ben formulated by the project team based on the preliminary data and literature precedent. For in vitro reconstitution, the project team will express and isolate each candidate biosynthetic enzyme. The strength of this approach is that it should unambiguously determine the co-substrates, cofactors, and products of each component enzyme, thereby chemically defining each step in the NNG biosynthetic pathway.

Future work on developing biocatalytic pathways to produce polynitramines will require studies of purified proteins; therefore, in vitro reconstitution allows the project team to simultaneously develop the enzyme isolation protocols for these anticipated efforts.

In completing this project, the team will identify each enzymatic step on the NNG biosynthetic pathway, which should, for the first time, completely elucidate the biosynthesis pathway for a nitramine-containing natural product. The project will also establish protocols anticipated to obtain high yields of the component biosynthetic proteins and unambiguously determine the co-substrates, cofactors, products of each component enzyme. These combined results are anticipated to directly enable further studies of the substrate scope or enable engineering efforts of the component proteins. The progression of this work will enable development of biocatalytic or synthetic biology strategies toward production of high-energy compounds relevant to Department of Defense. (Anticipated Project Completion - 2025)