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Interactive Learning Flavonoid Biosynthesis Preliminary remarks Hello and welcome to the interactive learning presentation „Flavonoid Biosynthesis“ . Please use the computer mouse to click through the presentation. For a simple handling, following pictograms are used to direct you through the presentation: Forward / Next slide Backward / Previous slide Back to selection Click on pulsating objects to gain more information And now: have fun! First, some biology ... Flavonoids ... ... are an important group of secondary metabolites ... have a wide range of physiological effects in us ... can be found in the whole plant kingdom Some examples for physiological functions in the plant Defense against herbivores UVb protection Antioxidative activity Control of growth processes Attracting birds Attracting pollinators Sun protection Metal complexation Radical scavenger Pollen fertility Antifreezing activity Communication with bacteria What‘s important for us? Anti-hepatatoxic effects Radical scavenger Cancer prevention Anti-viral effects Immune-modulatory effects Cytostatic effects Anti-edematous effects Protection against cardiovascular diseases Sugar transports Anti-oxidative compounds Anti-inflammatory effects Nearly the Same, but Different Flavan 4-ol Flavan 3-4-diol Flavan 3-ol Flavone Dihydroflavonol Isoflavone Anthocyanidin A C Flavonol B Flavanone The different flavonoid classes look quite similar, but are generally classified by the saturation and oxidation of the heterocycle C 3 Malonyl-CoA + 1 p-Coumaroyl CoA The General Flavonoid Pathway CHS Chalcone AUS Aurone CHI Flavanone FNS II Flavone FHT Dihydroflavonol DFR But don‘t worry! FLS Flavonol Leucoanthocyanidin ANS Anthocyanidin We‘ll do it together step by step! And all starts with ... The Amino Acid Phenylalanine • Formula: C9H11NO2 (165.19 g/mol) • Can be viewed as a benzyl group substituted for the methyl group of alanine • Synthezised via the shikimate pathway in plants and most microorganisms • An essential amino acid for animals • Used for the production of artificial sweetener Phenylalanine Ammonia Lyase What do you think? What will be removed by an ammonia lyase? NH3 .. H2O .. CH4 .. Phenylalanine Ammonia Lyase Phenylalanine • Formula: C9H8O2 (148.16 g/mol) • Melting Point: 133°C • Appears as white monoclinic crystals • Used in flavors, synthetic indigo and pharmaceuticals Cinnamic Acid Cinnamic acid Cinnamate 4-hydroxylase (C4H) p-Coumaric Acid Cinnamic Acid p-Coumaric Acid C4H p-Coumaric Acid gets in Touch with Cinnamate:Coenzyme A Ligase (4CL) What do you think? What will happen to p-coumaric acid? A carboxyl group will be added, resulting in Coenzyme A .. The p-coumaric acid will be cycled at the carboxyl group, resulting in Coenzyme A .. The Coenzyme A will be ligated to the p-coumaric acid, resulting in p-coumaroyl-CoA .. Formation of Chalcone: The First Step of Flavonoid Biosynthesis 3 Malonyl-CoA + 1 p-Coumaroyl CoA CHS Chalcone AUS Aurone CHI FNS II Flavanone Flavone FHT Dihydroflavonol FLS Flavonol DFR Leucoanthocyanidin ANS Anthocyanidin 1 x p-Coumaroyl-CoA + 3 x Malonyl-CoA The enzyme chalcone synthase (CHS) connects three acetate units derived from 3 malonylCoA decarboxylation reactions to the p-coumaroyl-CoA in condensation reactions p-Coumaroyl-CoA CHS CHS Finally, a new ring system is formed during a regiospecific C1,C6 Claison condenstation, generating the naringenin chalcone Naringenin chalcone CHS Chalcone: The First Fork in the Pathway Chalcone can further be processed to: Flavanone ... Anthocyanidin By Jubinrajop (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons Aurone Aurone Aurones are formed thanks to the enzyme aurone synthase (AUS). The precursors are chalcones. Aurones are an important group of yellow coloring pigments, besides carotenoids and some flavonols Prominent aurones are aureusidin and leptosidin What do you Think? What is Aureusidin Derived From? The biochemist Anton Aurueller, who described it first in 1943 .. The latin word „aureum“ for gold .. It‘s named after the brown algae Spatoglossum areullia .. Exactly! Aureusidin is a plant flavonoid that provides yellow coloration in several plants The pigments may have been developed to attract bees for pollination, but also provide protection from viruses and fungi 3 Malonyl-CoA + 1 p-Coumaroyl CoA Next step: Flavanone Formation CHS Chalcone AUS Aurone CHI FNS II Flavanone Flavone FHT Dihydroflavonol FLS Flavonol DFR Leucoanthocyanidin ANS Anthocyanidin The enzyme chalcone isomerase (CHI) catalyzes the formation of a new ring system via a Michael-type nucleophilic attack, generating the molecule naringenin Naringenin chalcone CHI F3‘H In addition, naringenin can be processed to eriodictyol thanks to the enzyme flavonoid 3‘hydroxylase (F3‘H). This will add a hydroxyl group at the C3‘ of the B-ring ... Naringenin can be processed in further steps of the flavonoid Naringenin biosynthesis, which we will see later F3‘5‘H Or, naringenin can be processed to pentahydroxyflavanone, thanks to flavonoid 3‘5‘-hydroxylase (F3‘5‘H). This will add 2 hydroxy groups at the C3‘ AND the C5‘ of the B-ring Naringenin Is the predominant flavanone in grapefruit, but can also be found in tomatoes and oranges Lowers cholesterol concentrations By Citrus_paradisi_(Grapefruit,_pink).jpg: (אAleph) derivative work: — raeky (Citrus_paradisi_(Grapefruit,_pink).jpg) [CC BY-SA 2.5 (http://creativecommons.org/licenses/by-sa/2.5)], via Wikimedia Commons Reduces hepatitis C virus production Is a precursor of pelargonidin (which we will see later) Next Step: Flavone Formation 3 Malonyl-CoA + 1 p-Coumaroyl CoA CHS Chalcone AUS Aurone CHI FNS II Flavanone Flavone FHT Dihydroflavonol FLS Flavonol DFR Leucoanthocyanidin ANS Anthocyanidin FNS II + NADPH + O2 Flavanone R1 = H; R2 = H Naringenin R1 = OH; R2 = H Eriodictyol R1 = OH; R2 = OH Pentahydroxyflavanone Flavone R1 = H; R2 = H Apigenin R1 = OH; R2 = H Luteolin R1 = OH; R2 = OH Tricetin There are two types of flavone synthases (FNS). Type I is oxoglutaratedependent and not very widespread in the plant kingdom. FNS II, however, has been observed in many plant species and is NADPH-dependent. Both introduce a double bond between C2 and C3 of the C-ring of flavanones. Next Step: Dihydroflavonol Formation 3 Malonyl-CoA + 1 p-Coumaroyl CoA CHS Chalcone AUS Aurone CHI FNS II Flavanone Flavone FHT Dihydroflavonol FLS Flavonol DFR Leucoanthocyanidin ANS Anthocyanidin FHT ... + Oxoglutarate + Fe(II) Flavanone R1 = H; R2 = H Naringenin R1 = OH; R2 = H Eriodictyol R1 = OH; R2 = OH Pentahydroxyflavanone Dihydroflavonol R1 = H; R2 = H Dihydrokaempferol R1= OH; R2 = H Dihydroquercetin R1 = OH; R2 = OH Dihydromyricetin The enzyme flavanone 3-hydroxylase (FHT) catalyzes the reaction from flavanone to dihydroflavonol. The reaction requires oxoglutarate and ferrous iron, whilst ascorbate stimulates the enzyme activity Quiztime!!! Did you pay attention? Whats the answer to this question? What are the cofactors of flavone synthase II? Oxoglutarat e and Iron (FE(II)) .. Ascorbate NADPH and oxygen .. .. Next Step: Flavonol Formation 3 Malonyl-CoA + 1 p-Coumaroyl CoA CHS Chalcone AUS Aurone CHI FNS II Flavanone Flavone FHT Dihydroflavonol FLS Flavonol DFR Leucoanthocyanidin ANS Anthocyanidin FLS + Oxoglutarate + Fe(II) + ascorbate Dihydroflavonol R1 = H; R2 = H Dihydrokaempferol R1= OH; R2 = H Dihydroquercetin R1 = OH; R2 = OH Dihydromyricetin Flavonol R1 = H; R2 = H Kaempferol R1= OH; R2 = H Quercetin R1 = OH; R2 = OH Myricetin Flavonol synthase (FLS) is an oxoglutarate-dependent enzyme, forming flavonols from dihydroflavonols by direct abstraction of the two vicinal hydrogen atoms in positions C2 and C3 of the C-ring, introducing a double bond. Cofactors are oxoglutarate, Fe(II) and ascorbate Next Step: Leucoanthocyanidin Formation 3 Malonyl-CoA + 1 p-Coumaroyl CoA CHS Chalcone AUS Aurone CHI FNS II Flavanone Flavone FHT Dihydroflavonol FLS Flavonol DFR Leucoanthocyanidin ANS Anthocyanidin DFR + NADPH Dihydroflavonol R1 = H; R2 = H Dihydrokaempferol R1= OH; R2 = H Dihydroquercetin R1 = OH; R2 = OH Dihydromyricetin Leucoanthocyanidin R1 = H; R2 = H Leucopelargonidin R1= OH; R2 = H Leucocyanidin R1 = OH; R2 = OH Leucodelphinidin Dihydroflavonol 4-reductase catalyzes the NADPH-dependent reduction of the carbonyl group of dihydroflavonols, resulting in leucoanthocyanidins, which are the immediate precursors of anthocyanidins Last, but not Least: The Formation of Anthocyanidin ? Anthocyanidin Leucoanthocyanidin Which enzyme will catalyze the reaction? Anthocyanidin Synthase (ANS) .. Anthocyanidin Reductase (ANR) .. Anthocyanidin Hydroxylase (ANH) .. ANS + oxoglutarate + ascorbate + Fe(II) Leucoanthocyanidin R1 = H; R2 = H Leucopelargonidin R1= OH; R2 = H Leucocyanidin R1 = OH; R2 = OH Leucodelphinidin Anthocyanidin R1 = H; R2 = H Pelargonidin R1= OH; R2 = H Cyanidin R1 = OH; R2 = OH Delphinidin Anthocyanidins are formed thanks to the enzyme anthocyanidin synthase (ANS). This oxoglutarate-dependent reaction introduces a double bond between C2 und C3 in the heterocycle C. Again, the reaction requires oxoglutarate, ascorbate and Fe(II). Just Small Details Lead to Big Differences!! Pelargonidin Cyanidin ... this leads to big differences in the phenotype! Though, the molecules differ only in the hydroxylation of the B-ring... Delphinidin Finally: Pelargonidin • Named after the plant genus Pelargonium Thanks to its light absorbance, it‘s bright red and can be found in... ... Pelargonium ... ... strawberries ... I, Prathyush Thomas [GFDL 1.2 (http://www.gnu.org/licenses/old-licenses/fdl-1.2.html) or FAL], via Wikimedia Commons ... or radish Finally: Cyanidin Thanks to its light absorbance, it appears dark red and can be found in... ... cherries ... ... plums ... By Evan-Amos (Own work) [CC0], via Wikimedia Commons ... or raspberries fir0002 | flagstaffotos.com.au [GFDL 1.2 (http://www.gnu.org/licenses/old-licenses/fdl-1.2.html)], via Finally: Delphinidin Thanks to its light absorbance, it‘s dark violet and can be found in... ... grapes ... fir0002 | flagstaffotos.com.au [GFDL 1.2 (http://www.gnu.org/licenses/old-licenses/fdl-1.2.html)], via ... Delphinium ... ... and eggplants By Horst Frank (Own work) [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], via Wikimedia Commons However, they‘re not done yet! There are other subsequent processes such as... And all for... There are many enzymes involved such as... Glycosylation Stabilization of the molecules Glucosyltransferase Recognition signal for transmembrane storage Acylation Methyltransferase Malonyltransferases Sulfuric ester formation Carboxylic ester formation Enhancement of water solubility Avoiding the interference with cellular membranes Sulfotransferases Methylation But for today, the formation of anthocyanidins should be enough I hope, you had fun and learned something today. Thank you and goodbye Picture Sources: • Cosmos: https://commons.wikimedia.org/wiki/File:Cosmos_sulphureus_%E0%B4%95%E0%B5%8B%E0%B4%B8%E0% B5%8D%E2%80%8C%E0%B4%AE%E0%B5%8B%E0%B4%B8%E0%B5%8D%E2%80%8C_%E0%B4%AA%E0%B5 %82%E0%B4%B5%E0%B5%8D%E2%80%8C.jpeg • Poinettia – Selecta One: http://www.selecta-one.com//media/filebase/images/produkte/03221_2.jpg • Coreopsis grandiflora – Selecta One: http://www.selectaone.com//media/filebase/images/produkte/03221_2.jpg • Grapefruit: https://commons.wikimedia.org/wiki/File:Citrus_paradisi_(Grapefruit,_pink)_white_bg.jpg • Pelargonium – Selecta One: http://www.selecta-one.com//media/filebase/images/produkte/04342_2.jpg • Strawberry: https://commons.wikimedia.org/wiki/File:Strawberry_BNC.jpg • Radish: https://commons.wikimedia.org/wiki/File:Remscheid_L%C3%BCttringhausen__Bauernmarkt_18_ies.jpg • Cherry: https://en.wikipedia.org/wiki/Cherry#/media/File:Black_Che.jpg • Plums: https://en.wikipedia.org/wiki/Plum#/media/File:Red-Plums.jpg • Raspberry: https://en.wikipedia.org/wiki/Raspberry#/media/File:Raspberries05.jpg • https://en.wikipedia.org/wiki/Grape#/media/File:Wine_grapes03.jpg • Delphinium: https://en.wikipedia.org/wiki/Delphinium#/media/File:Delphinium.jpg • Eggplant: https://de.wikipedia.org/wiki/Aubergine#/media/File:Aubergine.jpg I acknowledge funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 675657.
