Structural and mechanistic views of enzymatic, heme-dependent nitrogen-nitrogen bond formation

Molecules with nitrogen-nitrogen (N-N) bonds constitute a large group of clinically important drugs, and various synthetic approaches have been developed to construct functional groups like hydrazines, diazos, pyrazoles, and N-nitrosos. While hundreds of N-N-containing specialized natural metabolites have also been discovered, little is known about the underlying enzymatic mechanisms that have evolved for N-N bond formation. In order to directly form a single N(sp3)-N(sp3) bond, enzymes must reverse the typical nucleophilicity of one nitrogen. Here, we report structural and mechanistic interrogations of the piperazate synthase PipS, a heme-dependent enzyme that catalyzes an N-N bond forming cyclization of N5-OH-L-ornithine to give the non-proteinogenic amino acid L-piperazic acid. We show that PipS can process a variety of N-substituted hydroxylamines, to give either an imine or an N-N bond, in a substrate-specific manner. Using a combination of structural and biochemical experiments, computational studies, and spectroscopic characterization, we propose that heme-dependent dehydration and N-N bond formation in PipS proceed through divergent pathways, which may stem from a shared nitrenoid intermediate that effectively reverses the nucleophilicity of the hydroxylamine nitrogen. Our work expands the current knowledge of enzymatic N-N bond formation, and delineates the catalytic versatility of a heme cofactor, paving the way for future development of genetically encoded biocatalysts for N-N bond formation.