[P001] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P01-083] Comprehensive Genome Mining of ATP-Dependent Diazotases from Actinomycetes and Structural Basis of an Identified Promiscuous Diazotase

○Jiayu Ning¹, Seiji Kawai¹, Yohei Katsuyama^1,2, Yasuo Ohnishi^1,2 (1. Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo (Japan), 2. Collaborative Research Institute for Innovative Microbiology, The University of Tokyo (Japan))

Keywords:

Biosynthesis,Diazo group,Enzyme,Substrate specificity,Genome mining

The diazo group is a valuable functional group in synthetic and medicinal chemistry because of its broad synthetic reactivity. Although several diazotases that catalyze the condensation of an aromatic amine with nitrite using ATP have been identified in recent studies, their overall diversity, substrate scope, and the structural basis of their catalysis remain incompletely understood. In this study, we performed a comprehensive genome mining of ATP-dependent type I diazotases in actinomycetes. Sequence similarity network (SSN) analysis of putative AMP-binding enzymes encoded near creE and creD homologs for the ANS pathway, which synthesizes nitrite,¹ revealed two major families of diazotases, type Ia and type Ib. Because type Ia family diazotases were difficult to express and purify, we focused on the type Ib family. The type Ib family was further classified into 13 distinct groups using SSN analysis. Recombinant proteins of representative diazotases from multiple groups were prepared and evaluated in vitro using a library of aromatic amines. This evaluation revealed clear group-dependent differences in substrate specificity. Notably, diazotases in group 3 exhibited broad substrate specificities and consistently high catalytic activities across structurally diverse substrates.
We then selected a representative group 3 diazotase, Mco01_40450 from Microbispora corallina, for structural elucidation. To elucidate the structural basis of its activity, we determined its structure by cryo-electron microscopy single particle analysis (Cryo-EM-SPA) at 3.08 Å resolution. Mco01_40450 forms a dimer and binds AMP, pyrophosphate, and an aromatic amine substrate in its active site. A structural comparison with previously characterized diazotases⁶ revealed a significantly expanded substrate-binding pocket and a wider entrance region.
Structure-guided mutagenesis further demonstrated that both the size of the substrate-binding pocket and its accessibility determine substrate specificity. The substitution of bulky residues surrounding the pocket and its entrance enhanced activity toward sterically demanding aromatic amines. These results provide a rational framework for engineering diazotases with designed substrate profiles. These findings position group 3 diazotases as promising platforms for sustainable diazo synthesis and for developing biocatalysts for chemical biology and drug development.

References
1. Sugai, Y.; Katsuyama, Y.; Ohnishi, Y. Nat. Chem. Biol. 2016, 12, 73–75.
2. Kawai, S.; Hagihara, R.; Shin-Ya, K.; Katsuyama, Y.; Ohnishi, Y. Angew. Chem. Int. Ed. 2022, 61, e202211728.
3. Kawai, S.; Yamada, A.; Du, D.; Sugai, Y.; Katsuyama, Y.; Ohnishi, Y. ACS Chem. Biol. 2023, 18 (8), 1821–1828.
4. Kawai, S.; Yamada, A.; Katsuyama, Y.; Ohnishi, Y. Beilstein J. Org. Chem. 2024, 20, 1–11.
5. Kawai, S.; Ning, J.; Katsuyama, Y.; Ohnishi, Y. ChemBioChem 2024, e202400687.
6. Kawai, S.; Karasawa, M.; Moriwaki, Y.; Terada, T.; Katsuyama, Y.; Ohnishi, Y. Angew. Chem. Int. Ed. 2025, e202505851.