Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology-KAIST, Daejeon 305-701, Korea.
Biotechnol Bioeng. 2013 Feb;110(2):383-90. doi: 10.1002/bit.24729. Epub 2012 Oct 5.
Cytochrome P450 monooxygenases are multifunctional enzymes with potential applications in chemoenzymatic synthesis of complex chemicals as well as in studies of metabolism and xenobiotics. Widespread application of cytochrome P450s, however, is encumbered by the critical need for redox equivalents in their catalytic function. To overcome this limitation, we studied visible light-driven regeneration of NADPH for P450-catalyzed O-dealkylation reaction; we used eosin Y as a photosensitizing dye, triethanolamine as an electron donor, and [Cp*Rh(bpy)H₂O] as an electron mediator. We analyzed catalytic activity of cell-free synthesized P450 BM3 monooxygenase variant (Y51F/F87A, BM3m2) in the presence of key components for NADPH photoregeneration. The P450-catalyzed O-dealkylation reaction sustainably maintained its turnover with the continuous supply of photoregenerated NADPH. Visible light-driven, non-enzymatic NADPH regeneration provides a new route for efficient, sustainable utilization of P450 monooxygenases.
细胞色素 P450 单加氧酶是多功能酶，具有在复杂化学物质的化学酶促合成以及代谢和外源化学物质研究中的潜在应用。然而，细胞色素 P450 的广泛应用受到其催化功能中对氧化还原当量的关键需求的阻碍。为了克服这一限制，我们研究了可见光驱动的 NADPH 再生用于 P450 催化的 O-脱烷基化反应；我们使用曙红 Y 作为光敏染料，三乙醇胺作为电子供体，[Cp*Rh(bpy)H₂O]作为电子介体。我们分析了在关键的 NADPH 光再生组件存在下细胞游离合成的 P450 BM3 单加氧酶变体（Y51F/F87A，BM3m2）的催化活性。在持续供应光再生的 NADPH 的情况下，P450 催化的 O-脱烷基化反应可持续保持其周转率。可见光驱动的非酶 NADPH 再生为有效、可持续利用 P450 单加氧酶提供了新途径。
