Tea (Camellia sinensis) is an important commercial crop, in which the high content of flavonoids provides health benefits. A flavonoid glycosyltransferase (CsUGT73A20), belonging to cluster IIIa, was isolated from tea plant. The recombinant CsUGT73A20 in Escherichia coli exhibited a broad substrate tolerance toward multiple flavonoids. Among them, kaempferol was the optimal substrate compared to quercetin, myricetin, naringenin, apigenin, and kaempferide. However, no product was detected when UDP-galactose was used as the sugar donor. The reaction assay indicated that rCsUGT73A20 performed multisite glycosidation toward flavonol compounds, mainly forming 3-O-glucoside and 7-O-glucoside in vitro. The biochemical characterization analysis of CsUGT73A20 showed more K7G product accumulated at pH 8.0, but K3G was the main product at pH 9.0. Kinetic analysis demonstrated that high pH repressed the glycosylation reaction at the 7-OH site in vitro. Besides, the content of five flavonol-glucosides was increased in CsUGT73A20-overexpressing tobaccos (Nicotiana tabacum).
Tea (Camellia sinensis) is rich in flavan-3-ols (catechins), especially epicatechin (EC), which is the predominant extension unit of polymeric proanthocyanidins (PAs). However, studies assessing EC's stereochemistry are scarce. Here, a high performance liquid chromatography column using amylose tris-(3, 5-dimethylphenylcarbamate) immobilized on silica-gel as chiral stationary phases (CSPs) was applied to explore its stereochemistry and biosynthetic pathway in tea plants. The results revealed (-)-epicatechin [(-)-EC] was the predominant di-hyroxy-non-galloylated-catechins, while (+)-epicatechin [(+)-EC] was not detected. Interestingly, (-)-EC was the only product obtained from cyanidin using the partially purified native C. sinensis anthocyanidin reductase (CsANR) in the presence of reduction nicotinamide adenine dinucleotide phosphate (NADPH); meanwhile, (+)-EC was the main product using recombinant CsANR in the same conditions. In addition, (-)-EC could be obtained from (+)-catechin [(+)-C] using recombinant CsANR, which displayed C3-epimerase activity in the presence of oxidation nicotinamide adenine dinucleotide phosphate (NADP(+)). But the partially purified native CsANR did not possess this function. Finally, (-)-EC could result from the de-gallate acid reaction of epicatechin gallate (ECG) catalyzed by a novel partially purified native galloylated catechins hydrolase (GCH) from tea leaves. In summary, (-)-EC is likely the product of native protein from the tea plants, and (+)-EC is only produced in a reaction catalyzed by recombinant CsANR in vitro.
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