Functional analysis of flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylases from tea plant (Camellia sinensis), involved in the B-ring hydroxylation of flavonoids

Flavonoids are major polyphenol compounds in plant secondary metabolism. The hydroxylation pattern of the B-ring of flavonoids is determined by the flavonoid 3′-hydroxylase (F3’H) and flavonoid 3′,5′-hydroxylase (F3′5′H). In this paper, one CsF3′H and two CsF3′5′Hs (CsF3′5′Ha and CsF3′5′Hb) were isolated. The phylogenetic tree results showed that F3′H and F3′5′Hs belong to the CYP75B and CYP75A, respectively. The Expression pattern analysis showed that the expression of CsF3′5′Ha and CsF3′5′Hb in the bud and 1st leaf were higher than other tissues. However, the CsF3′H had the highest expression in the 4th and mature leaf. The correlation analysis showed that the expression of CsF3′5′Hs is positively associated with the concentration of B-trihydroxylated catechins, and the expression of CsF3′H is positively associated with the Q contentration. Heterologous expression of these genes in yeast showed that CsF3′H and CsF3′5′Ha can catalyze flavanones, flavonols and flavanonols to the corresponding 3′, 4′ or 3′, 4′, 5′-hydroxylated compounds, for which the optimum substrate is naringenin. The enzyme of CsF3′5′Hb can only catalyze flavonols (including K and Q) and flavanonols (DHK and DHQ), of which the highest activities in catalyzing are DHK. Interestingly, The experiment of site-directed mutagenesis suggested that two novel sites near the C-terminal were discovered impacting on the activity of the CsF3′5′H. These results provide a significantly molecular basis on the accumulation B-ring hydroxylation of flavonoids in tea plant.