Functional characterization of a Colchicum autumnale L. double-bond reductase (CaDBR1) in colchicine biosynthesis.

ABSTRACT

MAIN CONCLUSION: 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.