Image_3_Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in Camellia sinensis.jpeg (3.57 MB)

Image_3_Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in Camellia sinensis.jpeg

Download (3.57 MB)
figure
posted on 11.10.2019 by Keyi Huang, Ming Li, Yajun Liu, Mengqing Zhu, Guifu Zhao, Yihui Zhou, Lingjie Zhang, Yingling Wu, Xinlong Dai, Tao Xia, Liping Gao

Polyphenols play an important role in the astringent taste of tea [Camellia sinensis (L.)] infusions; catechins in phenolic compounds are beneficial to health. The biosynthesis of gallic acid (GA), a precursor for polyphenol synthesis, in tea plants remains unknown. It is well known that 3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key enzyme for catalyzing the conversion of 3-dehydroshikimate (3-DHS) to shikimate (SA); it also potentially participates in GA synthesis in a branch of the SA pathway. In this study, four CsDQD/SDH proteins were produced in Escherichia coli. Three CsDQD/SDHs had 3-DHS reduction and SA oxidation functions. Notably, three CsDQD/SDHs showed individual differences between the catalytic efficiency of 3-DHS reduction and SA oxidation; CsDQD/SDHa had higher catalytic efficiency for 3-DHS reduction than for SA oxidation, CsDQD/SDHd showed the opposite tendency, and CsDQD/SDHc had almost equal catalytic efficiency for 3-DHS reduction and SA oxidation. In vitro, GA was mainly generated from 3-DHS through nonenzymatic conversion. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that CsDQD/SDHc and CsDQD/SDHd expression was correlated with GA and 1-O-galloyl-β-D-glucose accumulation in C. sinensis. These results revealed the CsDQD/SDHc and CsDQD/SDHd genes are involved in GA synthesis. Finally, site-directed mutagenesis exhibited the mutation of residues Ser-338 and NRT to Gly and DI/LD in the SDH unit is the reason for the low activity of CsDQD/SDHb for 3-DHS reduction and SA oxidation.

History

References

Licence

Exports