MAIN CONCLUSION: In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3' position by flavonoid 3'-hydroxylase. The cytochrome P450-dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3' or 3' and 5', respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3'H and F3'5'H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3'H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3'-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3',5'-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3'Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.
MAIN CONCLUSION: In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3' position by flavonoid 3'-hydroxylase. The cytochrome P450-dependent monooxygenases flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3' or 3' and 5', respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3'H and F3'5'H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3'H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3'-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3',5'-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3'Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.
Authors: Jana Thill; Ionela Regos; Mohamed A Farag; Asma F Ahmad; Justyna Kusek; Ana Castro; Karin Schlangen; Christine Hayot Carbonero; Ilya Z Gadjev; Lydia M J Smith; Heidi Halbwirth; Dieter Treutter; Karl Stich Journal: Phytochemistry Date: 2012-07-18 Impact factor: 4.072
Authors: Daria Nitarska; Carmen Stefanini; Christian Haselmair-Gosch; Silvija Miosic; Benjamin Walliser; Maja Mikulic-Petkovsek; Ionela Regos; Ana Slatnar; Thomas Debener; Diro Terefe-Ayana; Vinicius Vilperte; Johannes Hadersdorfer; Karl Stich; Heidi Halbwirth Journal: BMC Plant Biol Date: 2018-10-03 Impact factor: 4.215
Authors: Mark Levisson; Constantinos Patinios; Sascha Hein; Philip A de Groot; Jean-Marc Daran; Robert D Hall; Stefan Martens; Jules Beekwilder Journal: Microb Cell Fact Date: 2018-07-03 Impact factor: 5.328