Structural and functional study suggests DnfC is a putative glutamine amidotransferase in the dirammox pathway

Microbial ammonia oxidation is essential for biogeochemical nitrogen cycling and wastewater treatment. Besides the well-studied nitrification and anaerobic ammonia oxidation, a novel ammonia oxidation process referred to as dir ect amm onia ox idation (dirammox) was recently discovered in heterotrophic nitrifier Alcaligenes members, where ammonia was converted to glutamine and oxidized to hydroxylamine and then to N 2 via a gene cluster, dnfABC . Two possible ammonia oxidation mechanisms were proposed, 1) glutamine is converted to some unknown compounds by potential glutamine amidotransferase DnfC and then oxidized to hydroxylamine by oxidase DnfAB, and 2) glutamine is oxidized to L-glutamic acid γ-hydroxamate (L-GlnγHXM) by DnfAB and then hydrolyzed to hydroxylamine by DnfC. Here, we determined the crystal structure of DnfC and identified a conserved catalytic pocket essential for hydroxylamine production and far larger than that required to accommodate a glutamate molecule. We found that the L-GlnγHXM hydrolysis activity is not necessary for hydroxylamine production in E. coli cells harboring dnfABC . Our structural and functional study of DnfC suggested that glutamine was converted to a so-far unknown compound and sequentially oxidized to hydroxylamine and N 2 . • DnfC structure reveals a class I glutamine amidotransferase fold. • A conserved catalytic pocket is essential for hydroxylamine production. • DnfC glutaminase activity is necessary but insufficient for dirammox. • L-GlnγHXM hydrolysis is not required for DnfABC-mediated NH 2 OH production.