Dataset Information

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

ABSTRACT: 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-?-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.

SUBMITTER: Huang K

PROVIDER: S-EPMC6797610 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in <i>Camellia sinensis</i>.

Huang Keyi K Li Ming M Liu Yajun Y Zhu Mengqing M Zhao Guifu G Zhou Yihui Y Zhang Lingjie L Wu Yingling Y Dai Xinlong X Xia Tao T Gao Liping L

Frontiers in plant science 20191011

Polyphenols play an important role in the astringent taste of tea [<i>Camellia sinensis</i> (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 synthes ...[more]

PMID: 31681371

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Project description:Main conclusionEucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B. The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a "classical" bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Dataset Information

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

Publications

Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in <i>Camellia sinensis</i>.

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