Real-time capture of domain movements during copper amine oxidase catalysis by mix-and-inject serial crystallography
Protein dynamics play a crucial role in various physiological functions, including enzyme catalysis. To explore conformational changes during enzyme catalysis, we conduct mix-and-inject serial crystallography, an advanced technique to capture time-resolved protein structures in real time, using the microcrystals of bacterial copper amine oxidase containing a protein-derived quinone cofactor. Within 50 ms of mixing the microcrystals (<4 μm) with a preferred substrate (2-phenylethylamine) under anaerobic conditions (reductive half-reaction), we observe domain movements associated with substrate binding and formation of a metastable reaction intermediate, a product Schiff-base of the quinone cofactor. At 100-1000 ms after mixing, conformational transition from aminoresorcinol to the semiquinone radical forms of the reduced cofactor progresses gradually, likely depending on the replacement of the product aldehyde by the next-cycle amine substrate that triggers the cofactor conformational change. Overall, this study provides structural insight into enzyme catalysis accompanying the active-site conformational changes that are hardly scrutinized by studies in solution.
Authors
- So Iwata (ORCID: https://orcid.org/0000-0003-1735-2937)
- I. Miyahara (ORCID: https://orcid.org/0000-0002-0968-0343)
- Jungmin Kang (ORCID: https://orcid.org/0000-0002-3506-6400)
- Eiichi Mizohata (ORCID: https://orcid.org/0000-0002-5526-5343)
- Katsuyuki Tanizawa
- Takehiko Tosha (ORCID: https://orcid.org/0000-0002-8971-0759)
- Taiki Murakami
- Eriko Nango (ORCID: https://orcid.org/0000-0001-9851-7355)
- Tomoyuki Tanaka (ORCID: https://orcid.org/0000-0002-9886-5947)
- T. Murakawa (ORCID: https://orcid.org/0000-0001-5905-532X)
- Fangjia Luo (ORCID: https://orcid.org/0000-0003-0631-6236)
- T. Sunami (ORCID: https://orcid.org/0000-0001-9155-5009)
- T. Yano (ORCID: https://orcid.org/0000-0002-9720-8339)
- Tetsuya Masuda (ORCID: https://orcid.org/0000-0001-5857-5753)
- Kensuke Tono (ORCID: https://orcid.org/0000-0003-1218-3759)
- Hisayuki Matsunaga
- Rie Tanaka (ORCID: https://orcid.org/0000-0003-1062-4792)
- Takehiro Higuchi
- Michihiro Sugahara
- Mamoru Suzuki (ORCID: https://orcid.org/0000-0001-7071-5093)
- Toshihide Okajima (ORCID: https://orcid.org/0000-0003-1733-9580)
- Kenji Fukui (ORCID: https://orcid.org/0000-0003-2463-7578)
- Tetsunari Kimura (ORCID: https://orcid.org/0000-0003-1650-7298)
- Teikoku Son
- Toshinori Oozeki
- Nobuyuki Nakajima
- K Sakai (ORCID: https://orcid.org/0000-0002-8247-4036)
- Toshi Arima
- Yoshiki Montawa
- Ikuya Kurauchi
Institutions
- Ryukoku University (JP)
- Osaka Gakuin University (JP)
- University of Hyogo (JP)
- Tohoku University (JP)
- Kyoto University (JP)
- SPring-8 (JP)
- Japan Synchrotron Radiation Research Institute (JP)
- National Institutes for Quantum Science and Technology (JP)
- Osaka Research Institute of Industrial Science and Technology (JP)
- Protein Research Foundation (JP)
- Osaka Metropolitan University (JP)
- Kobe University (JP)
- Osaka University of Pharmaceutical Sciences (JP)
- The University of Osaka (JP)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-12-18
- DOI
- https://doi.org/10.1038/s41467-025-67230-5
- Primary Topic
- Microbial metabolism and enzyme function
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Japan Agency for Medical Research and Development
- Ministry of Education, Culture, Sports, Science and Technology
- Japan Society for the Promotion of Science