Darimont Dominique, Weissenborn Martin J, Nebel Bernd A, Hauer Bernhard
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Leibniz-Institute of Plant Biochemistry (IPB), Weinberg 3, 06120 Halle (Saale), Germany; Martin-Luther-University Halle-Wittenberg, Institute of Chemistry, Germany.
Bioelectrochemistry. 2018 Feb;119:119-123. doi: 10.1016/j.bioelechem.2017.08.009. Epub 2017 Sep 18.
Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197-209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.
细胞色素P450酶的电化学体外还原是一种有前景的体内应用替代方法。之前我们展示了三种用于苯胺羟基化的工程化细胞色素P450变体，它们配备了碳纳米管结合肽（CNT标签），用于在CNT电极上自组装。与野生型细胞色素P450相比，其依赖于烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的活性有所增强，但偶联效率仍然较低。对于细胞色素P450 Verma，施瓦内贝格和罗卡塔诺（2014年，《生物聚合物》101卷，197 - 209页）通过分子动力学模拟计算了假定的电子转移途径（eTPs）。我们假设敲除这些转移途径会改变系统的偶联效率。结果表明，对于电驱动的细胞色素P450，没有改进的系统。然而，对于NADPH驱动的细胞色素P' 50，最活跃的eTP突变体在使用NADPH作为还原当量时，活性提高了13倍，偶联效率提高了32倍。这分别表明了NADPH和电极还原时电子传输的另一种原理。此外，这项工作还展示了一种提高细胞色素P450与非天然底物偶联和活性的工具。
