Abstract: | The formation of a N?N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N?N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate‐containing amino acid l ‐alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l ‐alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl‐carrier‐protein‐based mechanism for activation of the precursor l ‐diaminopropionate, and we also show that nitric oxide can participate in non‐enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N‐terminal cupin domain and a C‐terminal AraC‐like DNA‐binding domain, is required for alanosine biosynthesis. |