Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, Jiangsu, China.
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, Jiangsu, China.
Appl Microbiol Biotechnol. 2019 Jan;103(2):761-776. doi: 10.1007/s00253-018-9416-4. Epub 2018 Oct 27.
Tetrahydroprotoberberines (THPBs), a class of naturally occurring isoquinoline alkaloids, contain substituent methoxyl or hydroxyl groups which play a significant role in the pharmacological properties of these molecules. In this study, we report a biocatalytic strategy for selective O-demethylation of THPBs. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, exhibited markedly regioselective demethylation of nonhydroxyl-THPBs and monohydroxyl-THPBs on the D-ring. A possible binding mode of THPBs with CYP105D1 was investigated by docking analysis, and the results revealed that the D-rings of THPBs were with the minimum distance to the heme iron. Tetrahydropalmatine was used as a model substrate and enantioselective demethylation was demonstrated. (S)-Tetrahydropalmatine was only demethylated at C-10, while (R)-tetrahydropalmatine was first demethylated at C-10 and then subsequently demethylated at C-9. The kcat/Km value for demethylation of (R)-tetrahydropalmatine by CYP105D1 was 3.7 times greater than that for demethylation of (S)-tetrahydropalmatine. Furthermore, selective demethylation of (S)-tetrahydropalmatine by the CYP105D1-based whole-cell system was demonstrated for the highly efficient production of (S)-corydalmine which has distinct pharmacological applications, such as providing relief from bone cancer pain and reducing morphine tolerance. Moreover, a homologous redox partner was identified to enhance the catalytic efficiency of the CYP105D1-based whole-cell system. This is the first enzymatic characterization of a cytochrome P450 that has regio- and enantioselective demethylation activity of THPBs for application purpose. The cytochrome P450 system could be a promising strategy for selective demethylation in the pharmaceutical industry.
四氢原小檗碱（THPBs）是一类天然存在的异喹啉生物碱，含有取代的甲氧基或羟基，这些基团在这些分子的药理学性质中起着重要作用。在本研究中，我们报告了一种用于选择性 O-去甲基化 THPBs 的生物催化策略。来自链霉菌灰色亚种 ATCC 13273 的细胞色素 P450 CYP105D1 对 D-环上的非羟基-THPBs 和单羟基-THPBs 表现出明显的区域选择性去甲基化。通过对接分析研究了 THPBs 与 CYP105D1 的可能结合模式，结果表明 THPBs 的 D-环与血红素铁的距离最小。四氢巴马汀被用作模型底物并证明了对映选择性去甲基化。（S）-四氢巴马汀仅在 C-10 处被去甲基化，而（R）-四氢巴马汀首先在 C-10 处被去甲基化，然后在 C-9 处被去甲基化。CYP105D1 对（R）-四氢巴马汀去甲基化的 kcat/Km 值比（S）-四氢巴马汀去甲基化的 kcat/Km 值大 3.7 倍。此外，通过 CYP105D1 为基础的全细胞系统对（S）-四氢巴马汀进行选择性去甲基化，可高效生产具有明显药理学应用的（S）-刻叶紫堇碱，如缓解骨癌疼痛和减少吗啡耐受。此外，鉴定了同源的氧化还原伴侣以提高基于细胞色素 P450 的全细胞系统的催化效率。这是首次对具有 THPBs 区域和对映选择性去甲基化活性的细胞色素 P450 进行酶学表征，用于应用目的。细胞色素 P450 系统可能是制药行业中选择性去甲基化的有前途的策略。
