Cloning and functional characterization of the caffeine oxidase gene CsCDH from Camellia sinensis

Theacrine, a purine alkaloid with pharmacological effects such as calming and anti-depressive activities, is biosynthesized through a key rate-limiting enzyme, caffeine oxidase. Despite its importance, the caffeine oxidase gene (CsCDH) in Camellia sinensis has not been cloned to date. We successfully isolated the full-length CsCDH cDNA, which contains a 501-bp open reading frame (ORF) encoding a 166-amino-acid protein with a calculated molecular weight of 18.7 kDa. Molecular docking and dynamics simulations showed that CsCDH binds tightly and stably to caffeine, indicating its catalytic potential in converting caffeine to 1,3,7-trimethyluric acid. The CsCDH fusion protein was expressed in Escherichia coli and purified through affinity chromatography. In vitro enzymatic assays verified that CsCDH catalyzes the conversion of caffeine into 1,3,7-trimethyluric acid. Furthermore, transient expression in tobacco confirmed its caffeine oxidase activity in planta. Finally, antisense oligonucleotide (asODN) interference experiments confirmed that CsCDH exhibits caffeine oxidase activity in tea plants. This study lays the groundwork for unraveling the theacrine biosynthesis pathway and offers new insights into breeding low-caffeine or high-theacrine tea cultivars.