Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley.
Flavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e. the formation of apigenin from (2S)-naringenin. While many plants express a microsomal-type FNS II, the soluble FNS I appears to be confined to a few species of the Apiaceae and was cloned recently from parsley plants. FNS I belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenases characterized by short conserved sequence elements for cofactor binding, and its evolutionary context and mode of action are under investigation. Using a homology-based reverse transcription polymerase chain reaction approach, two additional flavonoid-specific dioxygenases were cloned from immature parsley leaflets, which were identified as flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS) after expression in yeast cells. Sequence alignments revealed marginal differences among the parsley FNS I and FHT polypeptides of only 6%, while much less identity (about 29%) was observed with the parsley FLS. Analogous to FNS I, FLS oxidizes the flavonoid gamma-pyrone by introducing a C2, C3 double bond, and (2R,3S)-dihydrokaempferol (cis-dihydrokaempferol) was proposed recently as the most likely intermediate in both FNS I and FLS catalysis. Incubation of either FNS I or FLS with cis-dihydrokaempferol exclusively produced kaempferol and confirmed the assumption that flavonol formation occurs via hydroxylation at C3 followed by dehydratation. However, the lack of apigenin in these incubations ruled out cis-dihydrokaempferol as a free intermediate in FNS I catalysis. Furthermore, neither (+)-trans-dihydrokaempferol nor unnatural (-)-trans-dihydrokaempferol and 2-hydroxynaringenin served as a substrate for FNS I. Overall, the data suggest that FNS I has evolved uniquely in some Apiaceae as a paraphyletic gene from FHT, irrespective of the fact that FNS I and FLS catalyze equivalent desaturation reactions.
Martens
Stefan
S
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17 A, Germany.
Forkmann
Gert
G
Britsch
Lothar
L
Wellmann
Frank
F
Matern
Ulrich
U
Lukacin
Richard
R
D000595:Amino Acid Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019143:Evolution, Molecular; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000494:pharmacology*; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D028528:Petroselinum / Q000201:enzymology*; D010802:Phylogeny; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C085313:flavonol synthase; D010100:Oxygen