Structure and mechanism of haem-dependent nitrogen–nitrogen bond formation in piperazate synthase

Molecules with nitrogen–nitrogen (N–N) bonds include diverse specialized metabolites from nature, but little is known about the underlying enzymatic mechanisms that have evolved for N–N bond formation. To directly form a single N(sp3)–N(sp3) bond, enzymes must reverse the typical nucleophilicity of one nitrogen. Here we report the structure of PipS, a haem-dependent enzyme that catalyses N–N bond formation in the cyclization of N5-OH-l-ornithine, giving l-piperazic acid. Our work reveals the role of a Lys–Thr dyad early in the mechanism and shows that PipS catalyses either N–N bond formation or imine-group formation in a substrate-specific manner, which may stem from a shared nitrenoid intermediate that effectively reverses the nucleophilicity of the hydroxylamine nitrogen. Our work expands knowledge of enzymatic N–N bond formation and delineates the catalytic versatility of a haem cofactor, paving the way for genetically encoded biocatalysts for N–N bond formation. Despite the existence of many N–N-containing natural metabolites, little is known about the enzymatic mechanisms of N–N bond formation. Now, a catalytically relevant X-ray crystal structure of an N–N-bond-forming enzyme, PipS, is reported and detailed insights into its catalytic mechanism are provided.