10.1021/jacs.8b13049.s001 Hai-Yan He Hai-Yan He Alyssa C. Henderson Alyssa C. Henderson Yi-Ling Du Yi-Ling Du Katherine S. Ryan Katherine S. Ryan Two-Enzyme Pathway Links l‑Arginine to Nitric Oxide in <i>N</i>‑Nitroso Biosynthesis American Chemical Society 2019 biosynthetic process biosynthetic reassembly l-arginine biosynthetic pathway biosynthetic gene cluster building chemical diversity urea compound role nonheme iron-dependent enzyme guanidino nitrogens nitroso formation Nitric Oxide understanding biosynthetic routes nitric oxide synthases nitroso group nitroso compound streptozocin enzyme pair N ω 2019-02-14 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Two-Enzyme_Pathway_Links_l_Arginine_to_Nitric_Oxide_in_i_N_i_Nitroso_Biosynthesis/7751951 Nitric oxide (NO) has wide-ranging roles in biology, but less is known about its role in building chemical diversity. Here we report a new route to NO from the biosynthetic pathway to the <i>N</i>-nitroso compound streptozocin. We show that the <i>N</i>-nitroso group of streptozocin comes from the biosynthetic reassembly of l-arginine, with the guanidino nitrogens forming a nitrogen–nitrogen bond. To understand this biosynthetic process, we identify the biosynthetic gene cluster of streptozocin and demonstrate that free l-arginine is <i>N</i>-methylated by StzE to give <i>N</i><sup>ω</sup>-monomethyl-l-arginine. We show that this product is then oxidized by StzF, a nonheme iron-dependent enzyme unrelated to known nitric oxide synthases, generating a urea compound and NO. Our work implies that formation and capture of NO is the likely route to <i>N</i>-nitroso formation in vivo. Altogether, our work unveils a new enzyme pair for the production of NO from l-arginine and sets the stage for understanding biosynthetic routes to <i>N</i>-nitroso natural products.