Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713AV, the Netherlands.
Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713AV, the Netherlands.
J Biotechnol. 2024 Jun 20;389:22-29. doi: 10.1016/j.jbiotec.2024.04.019. Epub 2024 Apr 30.
Rieske non-heme iron oxygenases (ROs) are redox enzymes essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful biocatalysts due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (HO) formation in the presence of a NADH cofactor regeneration system on the catalytic efficiency of CDO in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced HO formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h (TTN of 1515) at a 3-fold increase in production rate.
Rieske 非血红素铁加氧酶（ROs）是微生物生物降解和天然产物合成所必需的氧化还原酶。这些酶利用分子氧进行氧化反应，由于其广泛的反应范围和高选择性，因此它们是非常有用的生物催化剂。ROs 中氧的激活机制涉及相关蛋白组分的氧化还原中心之间的电子转移，形成电子转移链（ETC）。尽管 ETC 对于电子补充至关重要，但由于在氧激活过程中电子丢失，它会带来形成活性氧物种（ROS）的风险。我们之前的研究将 ROS 的形成与依赖黄素的三组分枯烯双加氧酶（CDO）中的黄素还原酶的 O 解偶联联系起来。在本研究中，我们通过在无细胞环境中研究 ROS 的形成对多组分 CDO 系统的影响来扩展这一发现。特别是，我们关注在存在 NADH 辅助因子再生系统的情况下，ROS 的形成对 CDO 体外催化效率的影响。基于此，我们提出了为 CDO 实施具有替代（非天然）氧化还原对的混合系统的方案，这在减少 HO 形成和增加产物形成方面具有很大的优势。由邻苯二甲酸二加氧酶（PDR）的 RO-还原酶和 CDO 组成的混合系统在茚的氧化官能化方面表现出最有前途的结果，在 3 倍的生产速率下，产物形成（20mM）在 24 小时内增加了 4 倍（TTN 为 1515）。
