AUTHOR=Huang Keyi , Li Ming , Liu Yajun , Zhu Mengqing , Zhao Guifu , Zhou Yihui , Zhang Lingjie , Wu Yingling , Dai Xinlong , Xia Tao , Gao Liping 

TITLE=Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in Camellia sinensis

JOURNAL=Frontiers in Plant Science

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2019.01268

DOI=10.3389/fpls.2019.01268

ISSN=1664-462X

ABSTRACT=<p>Polyphenols play an important role in the astringent taste of tea [<italic>Camellia sinensis</italic> (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 <italic>Cs</italic>DQD/SDH proteins were produced in <italic>Escherichia coli</italic>. Three <italic>Cs</italic>DQD/SDHs had 3-DHS reduction and SA oxidation functions. Notably, three <italic>Cs</italic>DQD/SDHs showed individual differences between the catalytic efficiency of 3-DHS reduction and SA oxidation; <italic>Cs</italic>DQD/SDHa had higher catalytic efficiency for 3-DHS reduction than for SA oxidation, <italic>Cs</italic>DQD/SDHd showed the opposite tendency, and <italic>Cs</italic>DQD/SDHc had almost equal catalytic efficiency for 3-DHS reduction and SA oxidation. <italic>In vitro</italic>, GA was mainly generated from 3-DHS through nonenzymatic conversion. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that <italic>CsDQD/SDHc</italic> and <italic>CsDQD/SDHd</italic> expression was correlated with GA and 1-<italic>O</italic>-galloyl-β-D-glucose accumulation in <italic>C. sinensis</italic>. These results revealed the <italic>Cs</italic>DQD/SDHc and <italic>Cs</italic>DQD/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 <italic>Cs</italic>DQD/SDHb for 3-DHS reduction and SA oxidation.</p>