Promiscuous ATP-Dependent Diazotases Discovered by Comprehensive Genome Mining Based on Sequence Similarity Network Analysis.
- 1Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo113-8657, Japan.
- 2Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo113-8657, Japan.
Abstract
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.
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BibTeX
@article{ning2026promiscuous,
title = {{Promiscuous ATP-Dependent Diazotases Discovered by Comprehensive Genome Mining Based on Sequence Similarity Network Analysis.}},
author = {Jiayu Ning and Seiji Kawai and Yohei Katsuyama and Yasuo Ohnishi},
journal = {Journal of the American Chemical Society},
year = {2026},
month = oct,
doi = {10.1021/jacs.6c11480},
url = {https://europepmc.org/article/MED/42913524},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Promiscuous ATP-Dependent Diazotases Discovered by Comprehensive Genome Mining Based on Sequence Similarity Network Analysis.
AU - Jiayu Ning
AU - Seiji Kawai
AU - Yohei Katsuyama
AU - Yasuo Ohnishi
PY - 2026
DA - 2026/10/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c11480
UR - https://europepmc.org/article/MED/42913524
AB - 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.
SN - 0002-7863
SN - 1520-5126
ER -