Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, China.
Hainan Key Laboratory of Sustainable Utilization of Tropical Bioresources, Key Laboratory for Quality Regulation of Tropical Horticultural Plants of Hainan Province, Sanya Nanfan Research Institute, College of Horticulture, Hainan University, Haikou, 570228, China.
Planta. 2022 Oct 10;256(5):95. doi: 10.1007/s00425-022-04003-0.
An alkenal double-bond reductase enzyme (CaDBR1) was cloned from Colchicum autumnale L. The encoded enzyme catalysed 4-coumaraldehyde to 4-hydroxydihydrocinnamaldehyde (4-HDCA). Its functional characterization increased the understanding of colchicine biosynthesis. As a traditional medical plant, Colchicum autumnale L. is famous for producing colchicine, a widely used drug for alleviating gout attacks. The biosynthetic pathway of colchicine was revealed most recently, and 4-hydroxydihydrocinnamaldehyde (4-HDCA) has been verified as a crucial intermediate derived from L-phenylalanine. However, the functional gene that catalyses the formation of 4-HDCA remains controversial. In this study, the alkenal double-bond reductase (DBR) gene member CaDBR1 was cloned and characterized from C. autumnale. Bioinformatics analysis predicted and characterized the basic physicochemical properties of CaDBR1. Recombinant CaDBR1 protein was heterologously expressed in Escherichia coli and purified by a Ni-NTA column. In vitro enzyme assays indicated that CaDBR1 could catalyse 4-coumaraldehyde to form 4-HDCA but could not generate 4-HDCA by taking cinnamaldehyde as a substrate. Stable transformation into tobacco BY-2 cells revealed that CaDBR1 localized in the cytoplasm, and tissue-specific expression results showed that CaDBR1 had the highest expression in bulbs. All these results verify and confirm the participation and contribution of CaDBR1 in the biosynthesis pathway of 4-HDCA and colchicine alkaloids in C. autumnale.
从秋水仙中克隆得到一种烯醛双键还原酶（CaDBR1）。该酶能催化 4-香豆醛生成 4-羟基肉桂醛（4-HDCA）。对该酶的功能表征增加了对秋水仙碱生物合成的理解。作为一种传统药用植物，秋水仙以产生秋水仙碱而闻名，秋水仙碱是一种广泛用于缓解痛风发作的药物。最近揭示了秋水仙碱的生物合成途径，并且已经验证 4-羟基肉桂醛（4-HDCA）是源自 L-苯丙氨酸的关键中间体。然而，催化 4-HDCA 形成的功能基因仍存在争议。在这项研究中，从秋水仙中克隆并表征了烯醛双键还原酶（DBR）基因成员 CaDBR1。生物信息学分析预测并表征了 CaDBR1 的基本理化性质。通过 Ni-NTA 柱异源表达重组 CaDBR1 蛋白并进行纯化。体外酶活性分析表明，CaDBR1 可以催化 4-香豆醛生成 4-HDCA，但不能以肉桂醛为底物生成 4-HDCA。稳定转化到烟草 BY-2 细胞表明 CaDBR1 定位于细胞质中，组织特异性表达结果表明 CaDBR1 在鳞茎中表达量最高。所有这些结果都证实和确认了 CaDBR1 在秋水仙烯醛双键还原酶生物合成途径中以及秋水仙碱类生物碱生物合成中的参与和贡献。
