Degregorio Danilo, D'Avino Serena, Castrignanò Silvia, Di Nardo Giovanna, Sadeghi Sheila J, Catucci Gianluca, Gilardi Gianfranco
Department of Life Sciences and Systems Biology, University of Turin Turin, Italy.
Front Pharmacol. 2017 Mar 21;8:121. doi: 10.3389/fphar.2017.00121. eCollection 2017.
Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a V increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.
人肝细胞色素P450 3A4是参与药物代谢的主要酶。这使其成为生物催化应用（如药物和药物代谢物的合成）的一个有吸引力的靶点。然而，其溶解性差、稳定性低以及偶联率低限制了其在生物技术领域的应用。我们之前证明，通过在来自BM3的还原酶（BMR）和N端修饰的P450 3A4（3A4-BMR）之间创建融合蛋白，可以提高P450 3A4的溶解性。在这项工作中，我们旨在通过改变连接两个结构域的环的长度来提高稳定性和偶联效率，以允许更高的结构域间灵活性，优化结构域之间的相互作用。从含有短接头Pro-Ser-Arg的构建体3A4-BMR开始，通过引入3个和5个甘氨酸铰链（3A4-3GLY-BMR和3A4-5GLY-BMR）生成了两个构建体。这三种融合蛋白在450 nm处显示出还原型血红素-CO加合物的典型吸光度，以及FAD和FMN辅因子的正确掺入。三种嵌合蛋白中的每一种都比单独的P450 3A4更稳定。此外，3A4-BMR-3-GLY酶显示出最高的NADPH氧化速率，这与最正的还原电位一致。另一方面，与3A4-BMR相比，3A4-BMR-5-GLY融合蛋白在标记底物睾酮的羟基化反应中显示出V增加了2倍以及更高的偶联效率。当使用这种外部电子受体从BMR向P450截获电子时，该蛋白还显示出细胞色素还原的最高速率值。数据表明，嵌合蛋白中结构域之间的灵活性和相互作用是提高周转率和偶联效率的关键参数。这些发现为工程化用于生物催化应用的催化自足型人P450提供了重要指导。
