The L-alanosine gene cluster encodes a pathway for diazeniumdiolate biosynthesis

N-nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein, we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product O_SCPCAPLC_SCPCAP-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the nitrogen source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid O_SCPCAPLC_SCPCAP-2,3-diaminopropionic acid (O_SCPCAPLC_SCPCAP-Dap) is synthesized and loaded onto a peptidyl carrier protein (PCP) domain in O_SCPCAPLC_SCPCAP-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. This research framework will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.