Characterizing Y224 conformational flexibility in FtmOx1-catalysis using <sup>19</sup>F NMR spectroscopy
α-Ketoglutarate-dependent non-haem iron (αKG-NHFe) enzymes play a crucial role in natural product biosynthesis, and in some cases exhibiting multifunctional catalysis capability. This study focuses on αKG-NHFe enzyme FtmOx1, which catalyzes endoperoxidation, dealkylation, and alcohol oxidation reactions in verruculogen biosynthesis. We explore the hypothesis that the conformational dynamics of the active site Y224 confer the multifunctional activities of FtmOx1-catalysis. Utilizing Y224-to-3,5-difluorotyrosine-substituted FtmOx1, produced <i>via</i> the amber codon suppression method, we conducted <sup>19</sup>F NMR characterization to investigate FtmOx1's structural flexibility. Subsequent biochemical and X-ray crystallographic analyses provided insights into how specific conformations of FtmOx1-substrate complexes influence their catalytic activities. These findings underscore the utility of <sup>19</sup>F NMR as a powerful tool for elucidating the complex mechanisms of multifunctional enzymes, offering potential avenues for developing biocatalytic processes to produce novel therapeutic agents harnessing their unique catalytic properties.
Authors
- Nathchar Naowarojna (ORCID: https://orcid.org/0000-0002-7952-3458)
- Shenlin Wang (ORCID: https://orcid.org/0000-0002-0153-0578)
- Pinghua Liu
- Xueting Liu (ORCID: https://orcid.org/0000-0002-1322-8253)
- Kelin Li (ORCID: https://orcid.org/0000-0002-8630-2928)
- Yu‐Chen Han (ORCID: https://orcid.org/0000-0001-9275-2039)
- Yi Ju (ORCID: https://orcid.org/0000-0003-2169-7970)
- Ke Ye (ORCID: https://orcid.org/0009-0003-9746-5452)
- Xinye Wang (ORCID: https://orcid.org/0000-0002-5776-7996)
- Wupeng Yan (ORCID: https://orcid.org/0000-0001-5387-6362)
- Lingyun Yang (ORCID: https://orcid.org/0000-0002-3186-3189)
- Li Xin Zhang (ORCID: https://orcid.org/0009-0003-7310-4468)
- Jun Wang (ORCID: https://orcid.org/0000-0002-7437-8503)
Institutions
- Boston University (US)
- East China University of Science and Technology (CN)
- Shanghai Jiao Tong University (CN)
- ShanghaiTech University (CN)
- Sakon Nakhon Rajabhat University (TH)
Publication Details
- Journal
- Catalysis Science & Technology
- Published
- 2024-11-20
- DOI
- https://doi.org/10.1039/d4cy01077a
- Citations
- 2
- Primary Topic
- Metal-Catalyzed Oxygenation Mechanisms
- Type
- article
- Field-Weighted Citation Impact
- 0.32
Funders
- National Science Foundation
- Salt Science Research Foundation
- National Natural Science Foundation of China
- State Key Laboratory of Bioreactor Engineering
- Higher Education Discipline Innovation Project
- National Institutes of Health
- National Key Research and Development Program of China
- Division of Chemistry