Discovery, Structure, and Mechanism of the (R, S)-Norcoclaurine Synthase for the Chiral Synthesis of Benzylisoquinoline Alkaloids,ACS Catalysis

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Discovery, Structure, and Mechanism of the (R, S)-Norcoclaurine Synthase for the Chiral Synthesis of Benzylisoquinoline Alkaloids
ACS Catalysis ( IF 11.3 ) Pub Date : 2023-11-08 , DOI: 10.1021/acscatal.3c03296
Libo Zhang ₁ , Shiqing Zhang _{2,

3} , Lijing Liao ₄ , Huanying Tang ₁ , Wenli Wang ₅ , Fucheng Yin ₆ , Liangliang Han ₆ , Kejin Zhu ₁ , Yushi Liu ₁ , Dingqiao Xu ₇ , Xiaobing Wang ₆ , Minjian Qin ₁ , Yibei Xiao ₄ , Xiang Sheng _{2,

3} , Yucheng Zhao ₁

Affiliation

Department of Resources Science of Traditional Chinese Medicines, School of Traditional Chinese Pharmacy, and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
National Center of Technology Innovation for Synthetic Biology and National Engineering Research Center of Industrial Enzymes, Tianjin 300308, China
Department of Pharmacology, School of Pharmacy, and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
School of Pharmacy, Nantong University, Nantong 226001, China
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
Key Laboratory of Shaanxi Administration of Traditional Chinese Medicine for TCM Compatibility, State Key Laboratory of Research & Development of Characteristic Qin Medicine Resources (Cultivation), Shaanxi Key Laboratory of Chinese Medicine Fundamentals and New Drugs Research, Shaanxi University of Chinese Medicine, Xi’an 712046, China

Benzylisoquinoline alkaloids (BIAs) are key plant metabolites that offer significant pharmacological benefits. While most naturally isolated BIAs are identified as (S)-enantiomers, (R)-configured BIAs are also abundant in specific species. However, the formation mechanism of (R)-enantiospecific BIAs remains largely unknown. Norcoclaurine synthase (NCS)-catalyzed Pictet–Spengler condensation is responsible for the BIAs scaffold formation and establishing a unique chiral carbon center. Nevertheless, all NCSs hitherto identified were strictly (S)-selective. Herein, five NCS homologues in lotus were identified and functionally characterized to be without enantiopreference, namely, being capable of producing (R)-norcoclaurine. We revealed the crystal structure of NnNCS1, one of the five identified NnNCS homologues, showing the enzyme’s typical pathogenesis-related protein 10-fold and an active dimeric form. Guided by the mechanistic information from quantum chemical calculations, the single-point mutation of Ile43, Leu60, and Phe101 leads to (R)-enantiospecific mutants. This study unravels the previously obscure pathway of (R)-BIA biosynthesis, thereby providing valuable enzymatic tools for the synthetic biology of (R)-configured BIAs.

中文翻译：

用于手性合成苄基异喹啉生物碱的 (R, S)-去甲月桂碱合酶的发现、结构和机制

苄基异喹啉生物碱 (BIA) 是重要的植物代谢物，具有显着的药理功效。虽然大多数天然分离的 BIA 被鉴定为 ( S )-对映体，但 ( R )-构型的 BIA 在特定物种中也很丰富。然而，( R )-对映特异性 BIA的形成机制仍然很大程度上未知。去甲乌云碱合酶 (NCS) 催化的 Pictet-Spengler 缩合负责 BIA 支架的形成并建立独特的手性碳中心。然而，迄今为止发现的所有 NCS 都是严格（S）选择性的。在此，鉴定了莲花中的五个NCS同源物，并进行了功能表征，其没有对映体优先性，即能够产生( R )-去甲乌云碱。我们揭示了Nn NCS1（五个已鉴定的Nn NCS 同源物之一）的晶体结构，显示了该酶典型的发病机制相关蛋白的 10 倍和活性二聚体形式。在量子化学计算的机制信息的指导下，Ile43、Leu60 和 Phe101 的单点突变导致 ( R )-对映特异性突变体。这项研究揭示了以前模糊的 ( R )-BIA 生物合成途径，从而为 ( R )-构型 BIA 的合成生物学提供了有价值的酶工具。

更新日期：2023-11-08

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