Promiscuous ATP-Dependent Diazotases Discovered by Comprehensive Genome Mining Based on Sequence Similarity Network Analysis.

The diazo group is a highly valuable functional group in organic synthesis; however, its conventional preparation requires harsh conditions. Here, we provide a systematic analysis of ATP-dependent diazotases from actinomycetes that catalyze the condensation of nitrite with aromatic amines. Sequence similarity network analysis of AMP-binding enzymes revealed two families (type Ia/Ib), with type Ib further classified into 14 groups. In vitro assays confirmed the diazotization activities of the diazotases from 7 groups in the type Ib family, each displaying distinct substrate preferences. Remarkably, diazotases in group 3 exhibited broad substrate specificity and superior catalytic efficiency. The dimeric structure of a representative enzyme, Mco01_40450, was determined at 3.08 Å resolution by cryogenic electron microscopy with C2 symmetry. Subsequent symmetry expansion and 3D classification revealed the structure with AMP, pyrophosphate, and 4-aminohydrocinnamic acid bound to the active site at a resolution of 3.16 Å. Expansion of the substrate-binding pocket and its entrance via amino acid substitution enabled the reaction of bulkier aromatic amines. Unlike classical chemical diazotization, Mco01_40450 operates under mild aqueous conditions and is compatible with sensitive protecting groups. Our findings highlight the structural and functional diversity of diazotases and establish group 3 diazotases as promising, engineerable biocatalysts for selective and efficient diazo installation.