Functional characterization of flavanone 3-hydroxylase gene from Phyllanthus emblica (L.)

Phyllanthus emblica (L.) is one of the important medicinal plants possessing antioxidant, anti-tumor, hepatoprotective and immune-modulatory properties imparted by presence of high amount of flavonoid and vitamin C contents. Despite the medicinal value of P. emblica, molecular characterization of genes such as those involved in biosynthesis of flavonoids is lacking. Flavanone 3-hydroxylase (F3H) [EC 1.14.11.9] is an important regulatory enzyme of the flavonoid pathway which catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydroflavonol. Herein, we isolated and investigated F3H gene from P. emblica, expressed functional recombinant proteins in microbial expression system and studied gene expression in different developmental stages. A full length PeF3H cDNA consisting of 1,469 bp (GenBank ID KF020698) has an open reading frame (ORF) of 1,095 bp, encoding a protein of 364 amino acids. Deduced amino acid analysis confirmed the presence of conserved amino acid residues which ligate ferrous iron and participates in 2-oxoglutarate binding (R-X-S). Three-dimensional protein structure modeling predicted that F3H had a jelly roll in the enzyme core, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including F3Hs. Phylogenetic analysis revealed that PeF3H was evolutionary closer F3H sequences of Triticum aestivum and Zea mays. Expression of PeF3H in an expression vector yielded a functional protein as HPLC analysis indicated conversion of naringenin to dihydrokaempferol after incubation with purified protein. Gene expression was analyzed in different developmental stages using q RT-PCR and was found to be highest in flowers as compared to leaves and fruits. In fruits, expression of PeF3H was increasing with the growth of the fruit.