Analysis of the spacial structure and active sites of caffeine synthase from Camellia ptilophylla chang

作者：Yan, Changyu; Ren, Qiujing; Chen, Xiaofang; Li, Bin; Chen, Zhongzheng^*

来源：Journal of Chinese Institute of Food Science and Technology, 2016, 16(9): 176-184.

DOI：10.16429/j.1009-7848.2016.09.024

• Accumulation effects
• Active site
• Camellia ptilophylla Chang
• Catalytic mechanisms
• Methyltransferases
• Spacial structures
• Substrate recognition
• Synthases

Caffeine synthase is the key enzyme which catalyze the caffeine synthesis in tea, but little is known on its spacial molecular structure which relation to its'catalytic mechanism. In this study, the cDNA library of Camellia ptilophylla Chang which owned high caffeine content was successfully constructed, and caffeine synthase genes were screened from the library and identified, then the spacial structure and the active site of the cloned caffeine synthase were analyzed by software Modeller 9.10 and Autodock 4.0. The main results showed a new caffeine synthase gene with 1 100 bp owned complete ORF and encoding 369 deduced amino acids was isolated and named as MYCS1, and the fusion protein which expressed from pET32a-MYCS1 in BL21 (DE3) shared the same catalytic activity as caffeine synthase. The spacial structure of MYCS1 indicated it owned nine α-helices and seven β-sheets, and including a globular domain with extended β-sheet and an active site cavity consist with a unique α-helical cap above the cavity. SAM as the methyl donor binding with MYCS1 was mediated through several hydrogen bond and the binding domain was composed of Asp63, Gly65, Asp103, Ser138, Phe139, Ser155. The methyl receptor including 7-mX and Tb binding with MYCS1 mainly the same as SAM by hydrogen bond formatted between them but affected by π-πaccumulation effect and strong hydrophobic effect which come from amino acid, and the binding domain mainly composed with Phe30, Thr31, Tyr156, Trp160 and Phe321. These results indicated that the spacial structure of caffeine synthase from Camellia ptilophylla Chang was similar to other SAM-dependent methyltransferases, and shared the similar conservative domain binding for substrate, but existed obviously difference in key domain for substrate recognition. The findings would be helpful for better understanding the caffeine synthase catalytic mechanism in tea plants.

• 出版日期2016
• 单位华南农业大学