Wang Xinye, Yang Lingyun, Wang Shenlin, Wang Jun, Li Kelin, Naowarojna Nathchar, Ju Yi, Ye Ke, Han Yuchen, Yan Wupeng, Liu Xueting, Zhang Lixin, Liu Pinghua
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology Shanghai 200237 China
iHuman Institute, Shanghaitech University Shanghai 201210 China.
Catal Sci Technol. 2024 Nov 20;15(2):386-395. doi: 10.1039/d4cy01077a. eCollection 2025 Jan 20.
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 the amber codon suppression method, we conducted 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 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.
α-酮戊二酸依赖的非血红素铁（αKG-NHFe）酶在天然产物生物合成中起关键作用，在某些情况下还具有多功能催化能力。本研究聚焦于αKG-NHFe酶FtmOx1，它在疣孢菌素生物合成中催化内过氧化物形成、脱烷基和醇氧化反应。我们探讨了活性位点Y224的构象动力学赋予FtmOx1催化多功能活性的假说。利用通过琥珀密码子抑制方法产生的Y224被3,5-二氟酪氨酸取代的FtmOx1，我们进行了F NMR表征以研究FtmOx1的结构灵活性。随后的生化和X射线晶体学分析深入了解了FtmOx1-底物复合物的特定构象如何影响其催化活性。这些发现强调了F NMR作为阐明多功能酶复杂机制的强大工具的实用性，为开发利用其独特催化特性生产新型治疗剂的生物催化过程提供了潜在途径。
