<i>N</i>-Hydroxylation and Hydrolysis by the DnfA/B/C Multienzyme System Involved in the Aerobic N<sub>2</sub> Formation Process
Lili Miao, Tingting Hou, Lan Ma, Min Wang, Ying Liu, Yao Wu, Yihua Chen, Wen-Zhao Wang, Ying Fu, Hua Xiang, Zhi‐Pei Liu
Abstract
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 acidγ-hydroxamate ( l -GlnγHXM), 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.