Unprecedented Nitrite‐Dependent Aromatic Amination to Synthesize 2,4‐Diamino‐3‐Hydroxybenzoic Acid

Aromatic rings bearing amino groups provide natural products with structural diversity and potent biological activities. Although aromatic amination is a useful reaction in organic synthesis, knowledge of biological aromatic amination remains limited. In this study, we identified an unprecedented nitrite-dependent aromatic amination in nybomycin biosynthesis. By comparing biosynthetic gene clusters whose products have a diamino phenol scaffold, we hypothesized that nine genes, including two nitrite biosynthetic genes, are responsible for the biosynthesis of this scaffold. Using heterologous expression in Streptomyces albus, we identified the minimum number of enzymes required for 2,4-diamino-3-hydroxybenzoic acid (2,4,3-DAHBA) biosynthesis. Further analysis revealed that three enzymes (NybN, NybO, and NybC) were responsible for converting 3-hydroxyanthranilic acid (3-HAA) into 2,4,3-DAHBA using nitrite. In vitro assays revealed that NybO, an ATP-dependent ligase, catalyzes the diazotization of 3-HAA to form 2-diazo-3-hydroxybenzoic acid (2,3-DHBA) and that NybC, an NADPH-dependent oxidoreductase, catalyzes the reduction of 2,3-DHBA to form 2-hydrazino-3-hydroxybenzoic acid. Taken together with other experimental results, we propose two possible biosynthetic pathways for 2,4,3-DAHBA synthesis from 3-HAA. This study provides important insights into nitrite-mediated aromatic amination, expanding the availability of nitrite for natural product biosynthesis.