LAQV-REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências Universidade do Porto, Rua do Campo Alegre, s/n, Porto 4169-007, Portugal.
J Phys Chem B. 2024 Oct 3;128(39):9447-9454. doi: 10.1021/acs.jpcb.4c05209. Epub 2024 Aug 26.
We have studied the hydroxylation mechanism of l-Tyr by the heme-dependent enzyme CYP76AD1 from the sugar beet (). This enzyme has a promising biotechnological application in modified yeast strains to produce medicinal alkaloids, an alternative to the traditional opium poppy harvest. A generative machine learning software based on AlphaFold was used to build the structure of CYP76AD1 since there are no structural data for this specific enzyme. After model validation, l-Tyr was docked in the active site of CYP76AD1 to assemble the reactive complex, whose catalytic distances remained stable throughout the 100 ns of MD simulation. Subsequent QM/MM calculations elucidated that l-Tyr hydroxylation occurs in two steps: hydrogen abstraction from l-Tyr by CpdI, forming an l-Tyr radical, and subsequent radical rebound, corresponding to a rate-limiting step of 16.0 kcal·mol. Our calculations suggest that the hydrogen abstraction step should occur in the doublet state, while the radical rebound should happen in the quartet state. The clarification of the reaction mechanism of CYP76AD1 provides insights into the rational optimization of the biosynthesis of alkaloids to eliminate the use of opium poppy.
我们研究了糖用甜菜来源的血红素依赖酶 CYP76AD1 对 l-Tyr 的羟化机制（）。该酶在通过改造酵母菌株生产药用生物碱方面具有广阔的生物技术应用前景，可替代传统的罂粟收获。由于没有该特定酶的结构数据，我们使用基于 AlphaFold 的生成式机器学习软件构建了 CYP76AD1 的结构。在模型验证后，l-Tyr 被对接至 CYP76AD1 的活性位点以组装反应复合物，在整个 100 ns 的 MD 模拟过程中，其催化距离保持稳定。随后的 QM/MM 计算阐明了 l-Tyr 的羟化作用分两步进行：CpdI 从 l-Tyr 中抽取氢，形成 l-Tyr 自由基，随后发生自由基反弹，对应于 16.0 kcal·mol 的限速步骤。我们的计算表明，氢提取步骤应发生在双重态，而自由基反弹应发生在四重态。阐明 CYP76AD1 的反应机制为合理优化生物碱的生物合成以消除罂粟的使用提供了思路。
