Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley

• Published in: FEBS letters 2003-06, Vol.544 (1), p.93-98
• Main Authors: Martens, Stefan, Forkmann, Gert, Britsch, Lothar, Wellmann, Frank, Matern, Ulrich, Lukačin, Richard
• Format: Article
• Language: English
• Subjects: 2-Oxoglutarate-dependent dioxygenase; Amino Acid Sequence; Apiaceae; Chromatography, Thin Layer; Cloning, Molecular; DNA, Complementary - metabolism; Evolution, Molecular; Flavonoid biosynthesis; Models, Chemical; Molecular Sequence Data; Oxidoreductases - chemistry; Oxidoreductases - pharmacology; Oxygen - metabolism; Peptides - chemistry; Petroselinum - enzymology; Petroselinum crispum; Phylogeny; Plant Proteins; Polymerase Chain Reaction; Recombinant Proteins - chemistry; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(03)00479-4

Flavone synthases (FNSs) catalyze the oxidation of flavanones to flavones, i.e. the formation of apigenin from (2 S)-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 3β-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 γ-pyrone by introducing a C2, C3 double bond, and (2 R,3 S)-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.