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 Cs F3′H and Cs F3′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 Cs F3′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 Cs F3′5′H. These results provide a significantly molecular basis on the accumulation B-ring hydroxylation of flavonoids in tea plant. Unlabelled Image • The CsF3 ′ H and CsF3 ′ 5 ′ Hs (CsF3 ′ 5 ′ Ha and CsF3 ′ 5 ′ Hb) genes related with B-ring of flavonoids were cloned from Camellia sinensis, and the function, optimum aubstrates, and key amino acid sites of these P450 enzymes were analysised in this paper. [ABSTRACT FROM AUTHOR]