Flavonoid-3',5'-hydroxylase from Phalaenopsis: a novel member of cytochrome P450s, its cDNA cloning, endogenous expression and molecular modeling.

A novel cDNA for the flavonoid-3',5'-hydroxylase (F35H) gene was cloned from petals of Phalaenopsis, and designated as Phf35h (accession number DQ148458 in GenBank/EMBL/DDBJ). The genomic clone of Phf35h was isolated by a PCR-based strategy. Nucleotide sequence analysis revealed that its genomic clone contains one intron and an open reading frame encoding a polypeptide of 507 amino acid residues. Southern hybridization analysis indicated the presence of a single gene coding for Phf35h. RT-PCR analysis showed that the Phf35h mRNA is transcribed in late phase of petal development, which is concomitant with the appearance of anthocyanins in petal tissue. The transcript is abundant in the purple petals but not in leaves or roots. The three-dimensional model of PhF3'5'H protein is classified into an alpha-domain which contains most of the alpha-helices with three small beta-sheets, a beta-domain that contains the larger beta-sheets with three small alpha-helices by homology modeling. The substrate-binding site for dihydrokaempferol on PhF3'5'H protein was determined by molecular docking algorithm. A highly conserved HPPTPLSLPH sequence was predicted to contact the aromatic ring of dihydrokaempferol.