Zhang Xuan, Wang Lanteng, Liu Jia, Chen Tzu-Yu, Yan Shengheng, Chang Wei-Chen, Shaik Sason, Zhou Jiahai, Wang Binju
State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China.
Institute of Drug Discovery Technology, Ningbo University, Ningbo 315211, China.
J Am Chem Soc. 2025 Jun 18;147(24):20442-20455. doi: 10.1021/jacs.5c02361. Epub 2025 Jun 6.
The C(sp)-C(sp) desaturation catalyzed by iron(II)- and 2-(oxo)glutarate-dependent(Fe/2OG) oxygenase is a key step in the biosynthesis and modification of natural products. Similar to other C-H functionalization processes, the reaction is initiated by the active Fe(IV)-oxo species, which abstracts a hydrogen atom from the C-H bond. However, Fe/2OG desaturase suppresses the thermodynamically favored OH-rebound process. This is enigmatic since the substrate-cofactor disposition appears to be a favorable process which involves C-H activation followed by OH rebound. To decipher the mechanism, we studied here the biosynthesis of dehydrofosmidomycin by DfmD, an Fe/2OG enzyme that catalyzes the biosynthesis of the natural product through desaturation, rearrangement, and demethylation reactions. This study employed biochemical, crystallographic, and computational analysis of the reaction. Unlike the sequential hydrogen-atom transfer (HAT) mechanism and cation-dependent mechanism, our study reveals an alternative mechanism for C-C desaturation. This mechanism involves the formation of a three-member ring intermediate oxaphosphiran. We found that the thermodynamically favored formation of oxaphosphiran reduced the barrier for the desaturation reaction. Additionally, the H-bonding network disfavors the OH-rebound pathway. As such, this dual action of the enzyme enables the selective desaturation reaction while bypassing the hydroxylation process. This mechanism highlights the importance of protein machinery as a means of controlling the reactivity and selectivity of radical species.
由铁（II）和2-（氧代）戊二酸依赖性（Fe/2OG）加氧酶催化的C（sp）-C（sp）去饱和反应是天然产物生物合成和修饰的关键步骤。与其他C-H官能化过程类似，该反应由活性Fe（IV）-氧物种引发，它从C-H键中提取一个氢原子。然而，Fe/2OG去饱和酶抑制了热力学上有利的OH-回弹过程。这很神秘，因为底物-辅因子的排列似乎是一个有利的过程，包括C-H活化，然后是OH回弹。为了解析其机制，我们在此研究了DfmD催化的脱氢磷霉素的生物合成，DfmD是一种Fe/2OG酶，通过去饱和、重排和脱甲基反应催化天然产物的生物合成。这项研究采用了该反应的生化、晶体学和计算分析。与顺序氢原子转移（HAT）机制和阳离子依赖性机制不同，我们的研究揭示了一种C-C去饱和的替代机制。该机制涉及形成一个三元环中间体氧杂磷环丙烷。我们发现，氧杂磷环丙烷在热力学上有利的形成降低了去饱和反应的势垒。此外，氢键网络不利于OH-回弹途径。因此，该酶的这种双重作用使得能够进行选择性去饱和反应，同时绕过羟基化过程。这种机制突出了蛋白质机制作为控制自由基物种反应性和选择性手段的重要性。
