Aix Marseille Univ, CNRS, Centrale Marseille, iSm2 UMR 7313, Marseille, France.
Delft University of Technology, The Netherlands.
Org Biomol Chem. 2021 Apr 26;19(15):3441-3450. doi: 10.1039/d1ob00015b.
Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.
双组分黄素蛋白单加氧酶由还原酶和加氧酶组成。后者在没有相应还原酶的情况下的功能证明以及黄素转移的机制仍然存在疑问。为了解决这个问题，我们利用无还原酶的反应系统，应用纯化的 2,5-二酮莰烷单加氧酶 I（2,5-DKCMO）、依赖黄素单核苷酸（FMN）的 II 型 Baeyer-Villiger 单加氧酶以及合成的烟酰胺类似物（NCBs）作为 FMN 还原的二氢吡啶衍生物。该系统证明了加氧酶的独立性能，以及 FMNH2 通过自由扩散进行运输的机制。该无还原酶系统的效率强烈依赖于 FMN 还原和酶（再）氧化的平衡，因为溶液中还原的 FMN 会导致不期望的副反应，例如过氧化氢的形成。实验设计使我们能够（i）研究各种反应参数的影响，强调平衡 FMN/FMNH2 循环的重要性，（ii）优化用于 rac-双环[3.2.0]庚-2-烯-6-酮、rac-樟脑和 rac-降樟脑的酶促 Baeyer-Villiger 氧化的反应体系。最后，这项研究不仅证明了 2,5-DKCMO 的还原酶独立性，而且还重新审视了这种特定情况下双组分黄素蛋白单加氧酶的术语。
