School of Pharmaceutical Sciences, Henan University of Chinese Medicine, No. 156 Jinshuidong Road, Zhengzhou, 450008, China; State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, NO.16 Neinanxiaojie, Dongcheng district, Beijing, China.
State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, NO.16 Neinanxiaojie, Dongcheng district, Beijing, China.
Plant Physiol Biochem. 2021 Nov;168:507-515. doi: 10.1016/j.plaphy.2021.09.042. Epub 2021 Oct 14.
Benzylisoquinoline alkaloids (BIAs) are compounds naturally found in plants and can have significant value in clinical settings. Metabolic engineering and synthetic biology are both promising approaches for the heterologous acquisition of benzylisoquinoline alkaloids. (S)-N-methylcoclaurine 3'-hydroxylase (NMCH), a member of the CYP80 family of CYP450, is the penultimate catalytic enzyme that forms the central branch-point intermediate (S)-reticuline and plays a key role in the biosynthesis of BIAs. In this study, an NMCH gene was cloned from Corydalis yanhusuo, while in vitro reactions demonstrated that CyNMCH can catalyze (S)-N-methylcoclaurine to produce (S)-3'-hydroxy-N-methylcoclaurine. The Km and Kcat of CyNMCH were estimated and compared with those identified in Eschscholzia californica and Coptis japonica. This newly discovered CyNMCH will provide alternative genetic resources for the synthetic biological production of benzylisoquinoline alkaloids and provides a foundation to help analyze the biosynthetic pathway of BIAs biosynthesis in C. yanhusuo.
苯并异喹啉生物碱（BIAs）是天然存在于植物中的化合物，在临床环境中具有重要价值。代谢工程和合成生物学都是异源获得苯并异喹啉生物碱的有前途的方法。（S）-N-甲基荷叶碱 3'-羟化酶（NMCH）是 CYP450 家族 CYP80 的成员，是形成中央分支点中间体（S）-reticuline 的最后一个催化酶，在 BIAs 的生物合成中起着关键作用。在这项研究中，从延胡索中克隆了 NMCH 基因，而体外反应表明 CyNMCH 可以催化（S）-N-甲基荷叶碱生成（S）-3'-羟基-N-甲基荷叶碱。估计了 CyNMCH 的 Km 和 Kcat，并与加利福尼亚罂粟和黄连中鉴定的 Km 和 Kcat 进行了比较。这种新发现的 CyNMCH 将为苯并异喹啉生物碱的合成生物学生产提供替代的遗传资源，并为分析 C. yanhusuo 中 BIAs 生物合成的生物合成途径提供基础。
