N-Hydroxylation and Hydrolysis by the DnfA/B/C Multienzyme System Involved in the Aerobic N₂ Formation Process

Nitrogen (N) loss is an important factor in N balance in ecosystems. Traditionally, ammonia was considered as the sole initial oxidizing substrate for N removal, and ammonia oxidation has long been a traditional N cycle topic. Dirammox is a distinctive nitrogen removal mechanism as it proceeds via direct aerobic conversion of ammonia to N-2 in a single bacterium, yet its biocatalytic reactions and enzymatic machinery remained elusive. By isotope tracing and biochemical experiments, here we elucidate the underlying reactions of N-oxidation [termed the dinitrogen-forming (DNF) pathway], which have little precedent in enzymology or organic amine utilization, expanding the catalytic capabilities of nonheme di-iron N-oxygenases (DnfA) and glutamine amidotransferases (GATase I, DnfC) to include N-2 formation. In the DNF pathway, amide N of glutamine is initially hydroxylated by DnfA with the assistance of DnfB to l-glutamic acid gamma-hydroxamate (l-Gln gamma HXM), which is then hydrolyzed by DnfC to hydroxylamine, which in turn is oxidized by DnfA/B to N-2. Further analyses revealed a wide distribution of N-hydroxylation-hydrolysis strategy for aerobic direct dinitrogen generation in various heterotrophic bacteria. These findings not only highlight the unusual organic amine N-hydroxylation reactions involved in N-3-oxidation but also provide insights into the universality of the DNF pathway and valuable biocatalysts in aerobic nitrogen removal.