A CYP80B enzyme from Stephania tetrandra enables the 3'-hydroxylation of N-methylcoclaurine and coclaurine in the biosynthesis of benzylisoquinoline alkaloids.
10.1016/S1875-5364(25)60867-3
- Author:
Yaoting LI
1
;
Yuhan FENG
2
;
Wan GUO
2
;
Yu GAO
3
;
Jiatao ZHANG
2
;
Lu YANG
4
;
Chun LEI
2
;
Yun KANG
2
;
Yaqin WANG
2
;
Xudong QU
5
;
Jianming HUANG
6
Author Information
1. School of Pharmacy, Fudan University, Shanghai 201203, China; State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
2. School of Pharmacy, Fudan University, Shanghai 201203, China.
3. School of Pharmacy, Fudan University, Shanghai 201203, China; School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
4. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
5. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: quxd19@sjtu.edu.cn.
6. School of Pharmacy, Fudan University, Shanghai 201203, China. Electronic address: jmhuang@shmu.edu.cn.
- Publication Type:Journal Article
- Keywords:
Benzylisoquinoline alkaloid;
CYP450 reductase;
CYP80B;
Cytochrome P450;
Stephania tetrandra
- MeSH:
Cytochrome P-450 Enzyme System/chemistry*;
Benzylisoquinolines/chemistry*;
Hydroxylation;
Plant Proteins/chemistry*;
Alkaloids/metabolism*;
Stephania tetrandra/genetics*
- From:
Chinese Journal of Natural Medicines (English Ed.)
2025;23(5):630-640
- CountryChina
- Language:English
-
Abstract:
Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
