Molecular characterization and functional expression of flavonol 6-hydroxylase.
BACKGROUND
Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.


RESULTS
A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome.


CONCLUSIONS
Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.
Anzellotti
Dominique
D
Molecular Membrane Dynamics Laboratory, Department of Anatomy and Cell Biology, McGill University, Montreal, H3A 2B2, Canada. dominique.anzellotti@mail.mcgill.ca
Ibrahim
Ragaï K
RK
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D018076:DNA, Complementary; D017343:Genes, Plant; D007438:Introns; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011794:Quercetin / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D031995:Saxifragaceae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
C073946:7,3',4'-trimethylquercetin; D003067:Coenzymes; D018076:DNA, Complementary; D011994:Recombinant Proteins; D011794:Quercetin; D006899:Mixed Function Oxygenases; C416048:flavonol 6-hydroxylase