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Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea

Citation

Dan, Hu et al. (2020), Identification and characterization of N9-methyltransferase involved in converting caffeine into non-stimulatory theacrine in tea, Dryad, Dataset, https://doi.org/10.5061/dryad.05qfttf05

Abstract

Caffeine is a major component of xanthine alkaloids and commonly consumed in many popular beverages. Due to its occasional side effects, reduction of caffeine in a natural way is of great importance and economic significance. Recent studies reveal that caffeine can be converted into non-stimulatory theacrine in the rare tea plant Camellia assamica var. kucha (Kucha), which involves oxidation at the C8 and methylation at the N9 positions of caffeine. However, the underlying molecular mechanism remains unclear. Here, we identify the theacrine synthase CkTcS from Kucha, which possesses novel N9-methyltransferase activity using 1,3,7-trimethyluric acid but not caffeine as a substrate, confirming that C8 oxidation takes place prior to N9-methylation. The crystal structure of the CkTcS complex reveals the key residues that are required for the N9-methylation, providing insights into how caffeine N-methyltransferases in tea plants have evolved to catalyze regioselective N-methylation through fine tuning of their active sites. These results may guide the future development of decaffeinated drinks.

Methods

Transcriptome sequencing performed at the Novogene Bioinformatics Institute (Novogene, Beijing, China). Briefly, mRNA was purified from the total RNA of tea leaves (Camellia assamica var. kucha and Camellia sinensis var.assamica) and used to construct sequencing libraries using a NEBNext® Ultra RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s recommendations. The index codes were added to attribute sequences to each sample. After the qualities of sequencing libraries were confirmed on the Agilent Bioanalyzer 2100 system, clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA) according to the manufacturer’s instructions. The library preparations were then sequenced on an Illumina Hiseq 2500 platform (Illumina, San Diego, CA, USA) and paired-end reads were generated. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity with min_kmer_cov set to 2 by default and all other parameters set default.

Funding

National Natural Science Foundation of China, Award: 81622050