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TitleStructural Basis for the Catalytic Mechanism of ATP-Dependent Diazotase CmaA6.
Journal, issue, pagesAngew Chem Int Ed Engl, Vol. 64, Issue 27, Page e202505851, Year 2025
Publish dateMay 5, 2025
AuthorsSeiji Kawai / Masayuki Karasawa / Yoshitaka Moriwaki / Tohru Terada / Yohei Katsuyama / Yasuo Ohnishi /
PubMed AbstractAlthough several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP-dependent ...Although several diazotases have been recently reported, the details of the reaction mechanism are not yet understood. In this study, we investigated the mechanism of CmaA6, an ATP-dependent diazotase, which catalyzes the diazotization of 3-aminocoumaric acid using nitrous acid. X-ray crystallography and cryogenic electron microscopy-single particle analysis revealed CmaA6 structures in the substrate-free and AMP-binding states. Kinetic analysis suggested that CmaA6 catalyzes diazotization via a sequential reaction mechanism in which three substrates (nitrous acid, ATP, and 3-aminocoumaric acid) are simultaneously bound in the reaction pocket. The nitrous acid and 3-aminocoumaric acid binding sites were predicted based on the AMP-binding state and confirmed by site-directed mutagenesis. In addition, computational analysis revealed a tunnel for 3-aminocoumaric acid to enter the reaction pocket, which was advantageous for the sequential reaction mechanism. This study provides important insights into the catalytic mechanism of diazotization in natural product biosynthesis.
External linksAngew Chem Int Ed Engl / PubMed:40275441 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution2.73 - 3.48 Å
Structure data

EMDB-61363: Structure of ATP-dependent diazotase, CmaA6
Method: EM (single particle) / Resolution: 3.48 Å

PDB-8ztz:
Structure of ATP-dependent diazotase CmaA6
Method: X-RAY DIFFRACTION / Resolution: 2.93 Å

PDB-9ijf:
Structure of ATP-dependent diazotase CmaA6
Method: X-RAY DIFFRACTION / Resolution: 2.73 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

ChemComp-HOH:
WATER

Source
  • kutzneria albida dsm 43870 (bacteria)
KeywordsLIGASE / enzyme / diazotase

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