Molecules with a nitrogen-nitrogen (N-N) bond in their structures exhibit various biological activities and other unique properties. A few microbial proteins are recently emerging as dedicated N-N bond forming enzymes in natural product biosynthesis. However, the details of these biochemical processes remain largely unknown. Here, through in vitro biochemical characterization and computational studies, we report the molecular basis of hydrazine bond formation by a family of di-domain enzymes. These enzymes are widespread in bacteria and sometimes naturally exist as two standalone enzymes. We reveal that the methionyl-tRNA synthase-like domain/protein catalyzes ATP-dependent condensation of two amino acids substrates to form a highly unstable ester intermediate, which is subsequently captured by the zinc-binding cupin domain/protein and undergoes redox-neutral intramolecular rearrangement to give the N-N bond containing product. These results provide important mechanistic insights into enzymatic N-N bond formation and should facilitate future development of novel N-N forming biocatalyst.

Pyrazomycin is a rare C‐nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that the StrR‐family regulator PyrR is the cluster‐situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable‐isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen–nitrogen bond‐forming enzyme that catalyzes the linkage of the ϵ‐NH2 nitrogen atom of l‐N6‐OH‐lysine and the α‐NH2 nitrogen atom of l‐glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.

The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.

Luzopeptins and related decadepsipeptides are bisintercalator nonribosomal peptides featuring rare acylsubstituted tetrahydropyridazine-3-carboxylic acid (Thp) subunits that are critical to their biological activities.H erein, we reconstitute the biosynthetic tailoring pathwayinluzopeptin A biosynthesis through in vivo genetic and in vitro biochemical approaches.S ignificantly,w er evealed am ultitasking cytochrome P450 enzyme that catalyzesf our consecutive oxidations including the highly unusual carbon-nitrogen bond desaturation, forming the hydrazone-bearing 4-OH-Thp residues.M oreover,w ei dentified am embrane-bound acyltransferase that likely mediates the subsequent O-acetylation extracellularly,a sapotential self-protective strategy for the producer strain. Further genome mining of novel decadepsipeptides and an associated P450 enzyme have provided mechanistic insights into the P450-mediated carbon-nitrogen bond desaturation. Our results not only reveal the molecular basis of pharmacophore formation in bisintercalator decadepsipeptides,b ut also expand the catalytic versatility of P450 family enzymes.

The atmospheric oxidation of chemicals has produced many new unpredicted pollutants. A microwave plasma torch-based ion/molecular reactor (MPTIR) interfacing an online mass spectrometer has been developed for creating and monitoring rapid oxidation reactions. Oxygen in the air is activated by the plasma into highly reactive oxygen radicals, thereby achieving oxidation of thioethers, alcohols, and various environmental pollutants on a millisecond scale without the addition of external oxidants or catalysts (6 orders of magnitude faster than bulk). The direct and real-time oxidation products of polycyclic aromatic hydrocarbons and p-phenylenediamines from the MPTIR match those of the long-term multistep environmental oxidative process. Meanwhile, two unreported environmental compounds were identified with an MPTIR and measured in the actual water samples, which demonstrates the considerable significance of the proposed device for both predicting the environmental pollutants (non-target screening) and studying the mechanism of atmospheric oxidative processes.