Transcript Flavanoid-Biosynthesis

Interactive Learning
Flavonoid Biosynthesis
Preliminary remarks
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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.