| Flavonoids comprise the most common group of polyphenolic plant secondary metabolites. There are diverse group of plants containing flavone, isoflavone, flavonol, chalcone, etc. They play key roles in biological activities. Flavonols are an important part of flavonoids and produced by flavonol synthase.According to the conserved cDNA sequence of flavonol synthase(FLS) genes, a pair of primers was designed,and flavonol synthase gene was cloned from tobacco (Nicotiana tabacun L.) leaves using RT-PCR and RACE methods. The full length cDNA sequence was 1186bp and included a whole ORF,which encoded 333 residues. The amino acid identity among FLSs from different species was high, ranging from 47% to 87%. The enzyme belongs to the class 2-oxoglutarate-dependent dioxygenases. Some similar domains to Isopenicillin II synthase and 2OG-Fe(II) oxygenase superfamily were found when searching the protein conserved domains in GenBank. Polypeptide alignments of FLSs with 2-oxoglutarate-dependent dioxygenases and related 2-oxoglutarate-independent enzymes revealed eight strictly conserved amino acids in three regions. Homology modeling was used to predict the tertiary structure. The results of homology modeling indicated that the main chain of FLS contained 11β-strands, of which formed a -jellyroll or double-strandedβhelix topology. The jellyroll formed a hydrophobic cavity, one end of which formed the active site. Four strictly conserved amino acids (His219, His275, Asp221, Arg285) were in the hydrophobic cavity and others (Gly65, His72, Pro205, Gly259) in turn. Like the other dioxygenases of this class, flavonol synthase cDNA encoded eight strictly conserved amino-acid residues which included two histidines (His219, His275) and one acidic amino acid (Asp221) residue for Fe(II)-coordination, an arginine (Arg285) proposed to bind 2-oxoglutarate, and the rest four (Gly65, His72, Pro205, Gly259) required for proper folding of the flavonol synthase polypeptide.
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