The Biosynthetic Gene Cluster of Pyrazomycin—A C‐Nucleoside Antibiotic with a Rare Pyrazole Moiety

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. Connecting the nitrogen atoms: Identification of the pyrazomycin biosynthetic gene cluster from S. candidus NRRL 3601 and its subsequent in vivo reconstitution has enabled the discovery of PyrN, a new zinc‐binding enzyme that catalyzes nitrogen–nitrogen bond formation and is responsible for linking the two nitrogen atoms in pyrazomycin's unusual pyrazole ring.