MPS587 - Advanced Plant Biochemistry Course Fall Semester 2011

MPS587 - Advanced Plant Biochemistry Course
Fall Semester 2011
Lecture 8
Phenylpropanoids IV
1. Flavonoids and related phenylpropanoids - overview
2. Flavonoids and anthocyanins
3. Phlobaphenes and proanthocyanidins
4. Isoflavonoids
5. Focus: O-methyltransferases
6. Focus: glycosyltransferases
Flavonoids and related
phenylpropanoid-acetate
metabolites –
biosynthetic overview
(Winkel-Shirley (2001)
Plant Physiology 126: 485)
2
Flavonoid nomenclature
3
Functional roles of flavonoids
(Pollastri & Tattini (2011) Annals of Botany doi: 10.1093/aob/mcr234)
Quercetin derivatives in the
nano- to micromolar range may
regulate both the cellular redox
homeostasis and developmental
processes. They may inhibit the
phosphorylation of auxin efflux
facilitator proteins located at
both the endoplasmic reticulum
(ER) and the plasma membrane
(PM). The presence of the whole
set of genes for quercetin
biosynthesis, coupled with the
occurrence of ‘short’ PIN
proteins at the ER (the site of
flavonoid biosynthesis) detected
in liverworts and mosses,
suggests ancestral functions for
flavonols as developmental
regulators. Quercetin derivatives
have also been shown to tightly
control the oxidative stressinduced MAPK activities in
animals, but conclusive evidence
for this functional role in plants
is still lacking (dotted arrows at
the bottom).
4
Flavonoids as
Benzalacetone in rhubarb
(Morita
et al. (2010) Proceedings of the National Academy of Sciences in the USA 107: 669)
developmental
regulators
(Taylor & Grotewold (2005) Current
Opinion in Plant Biology 8: 317)
5
Core flavonoid/anthocyanidin pathway
(Grotewold (2006) Annual Reviews in Plant Biology 57: 761-780)
Schematic representation of the
biosynthetic pathway of the most
abundant anthocyanin pigments.
The names of the compounds are
indicated. The enzyme names, in
black boxes, are CHS, chalcone
synthase;
CHI,
chalcone
isomerase; F3H, flavanone 3hydroxylase; F3′H, flavanone 3′hydroxylase; F3′5′H, flavanone
3′,5′-hydroxylase;
DFR,
dihydroflavonol
4-reductase;
LDOX/ANS,
leucoanthocyanidin
dioxygenase/anthocyanidin
synthase. The A-, B-, and C-rings
with the carbon numbers are
indicated in the structure
corresponding to the flavanone
naringenin.
6
Chalcone isomerase
(Jez & Noel (2002) Journal of Biological Chemistry 277: 1361)
CHI-catalyzed reaction and active site
architecture.
A, overall reaction catalyzed by CHI.
B, view of the active site hydrogen bond
network in the CHI·naringenin complex
(17). Hydrogen bond interactions (small
spheres) occur within a network centered
on two water molecules (red spheres) that
contact the flavanone ketone oxygen and
through interactions of the 7-hydroxyl
moiety of the flavanone product with
Asn113 and Thr190.
C, proposed cyclization reaction catalyzed
by CHI. After nucleophilic attack of the 2′oxyanion on the α,β-unsaturated double
bond, a water molecule acts as a general
acid to stabilize the enolate, resulting in
formation of a flav-3-en-4-ol intermediate
that tautomerizes into the expected
reaction product.
7
Flavanone 3β-hydroxylase and dihydroflavonol reductase
(Springob et al. (2003) Natural Products Reports 20: 288)
Flavanones are converted to dihydroflavonols by a
hydroxylation in position 3 catalyzed by flavanone 3βhydroxylase (F3H). This enzyme is classified as a soluble 2oxoglutarate-dependent dioxygenase according to its
requirement of the co-factors 2-oxoglutarate, molecular
oxygen, ferrous iron (Fe(II)) and ascorbate. F3H catalyzes the
stereospecific hydroxylation of (2S)-naringenin and (2S)eriodictyol to form (2R,3R)-dihydrokaempferol and (2R,3R)dihydroquercetin, respectively.
DFR catalyzes the stereospecific conversion of (2R,3R)dihydroflavonols to (2R,3S,4S)-leucoanthocyanidins DFR
requires NADPH as reducing cofactor and catalyzes the
transfer of the pro-S hydrogen of NADPH to the re-face of
the 4-keto group of dihydroflavonol.
8
Anthocyanidin synthase is a oxoglutarate-dependent dioxygenase
(Wilmouth et al. (2002) Structure 10: 93)
Proposed mechanism for anthocyanidin synthase
catalyzed C-3 hydroxylation, with the oxidation of
a substrate analogue, dihydroquercetin (DHQ), to
quercetin shown as an example. The scheme is
meant to indicate preferred pathways only. It is
proposed that the hydroxylation step during
oxidation of the leucoanthocyanidins follows a
similar catalytic scheme. The DHQ substrate is
shown in blue, 2OG is in green, and the oxygen
atoms from dioxygen are in purple.
9
Flower colors from anthocyanins
(Tanaka et al. (2009) The Plant Journal 54: 733)
(a) Gentian flowers containing diacylated delphinidin-based anthocyanins, such as gentiodelphin (right). Glucosylation (3′GT, 5GT, 3′GT) and
acylation (5,3′AT) reactions are indicated by arrows. (b) Chrysanthemum petals contain cyanidin 3-(6″-malonyl or 3″,6″-dimalonyl)-glucoside.
3MAT1, malonyl CoA:anthocyanin 3-malonyltransferase; 3MAT2, malonyl CoA: anthocyanin 3-dimalonyltransferase. (c) A blue poppy,
Meconopsis horridula. The sky-blue color of Meconopsis petals depends on cyanidin-based anthocyanin, flavonol and Fe3+. (d) Strongylodon
macrobotrys, a bat-pollinated plant of the Philippines, with unusual bluish-green flowers. They contain malvidin 3,5-diglucoside and flavones.
An uncharacterized factor seems to yield the greenish color. (e) The leaves of red Perilla are a source of a natural colorant containing
cyanidin. A Perilla anthocyanin is shown. 3AT, hydroxycinnamoyl CoA:anthocyanin 3-hydroxycinnamoyltransferase 5MAT, malonyl CoA:
anthocyanin 5-malonyltransferase.
10
Proanthocyanidins - chemistry
(Ferreira & Slade (2002) Natural Products Reports 19: 517)
11
Proanthocyanidins
(Jez & Yu (year) The Plant Journal
Vol: Page)
(a) The flavonoid biosynthetic enzymes
provide naringenin for catechin production
(green). A series of enzyme-catalyzed reactions
(purple) leads to the generation of catechin
and epicatechins. The stereochemistry of the
hydroxyl group in red is determined by the
preceding enzymatic reactions.
(b) Chemical structures of catechin epimers
(i.e. (+)-catechin and (−)-epicatechin) and
catechin derivatives (EGC and EGCG).
12
Leucoanthocyanidin reductase
(Maugé et al. (2010) Journal of Molecular Biology 397: 1079)
Ordering of the VvLAR1 171–175 fragment in the vicinity of the binding site.
(a) Solvent-accessibility surface of the substrate binding site as observed in the binary complex I structure. Red spheres
represent water molecules. The NADPH molecule is represented using a stick model (carbon atoms are colored green).
(b) Helix 3b interactions with product and coenzyme. The mFo − DFc electron density omit map is contoured at 2.5 σ level
with NADPH, (+)-catechin, and residues 171–175 excluded from the model. The map contouring residues 171 to 175,
which could only be modeled in the ternary complex structure, is colored dark blue. The (+)-catechin molecule is
represented using a stick model (carbon atoms are colored pink).
(c) Solvent-accessibility surface in the ternary complex structure [same orientation as in (a) and same color code as in (a)
and (b)].
13
Leucoanthocyanidin reductase
(Maugé et al. (2010) Journal of
Molecular Biology 397: 1079)
Hydrogen-bonding network in VvLAR1
active site and proposed reaction
mechanism. (a) VvLAR1 active site and its
interaction with NADPH and (+)-catechin.
Hydrogen bonds are shown in broken
lines; NADPH is represented in green and
(+)-catechin is represented in pink. (b)
Catalytic mechanism proposed for
VvLAR1. B stands for Lys140, which
promotes
the
substrate
OH7
deprotonation via Wat1. A stands for
His122, which is hydrogen-bonded to
Wat2.
14
Regulation of flavonoid/proanthocyanidin biosynthesis
Broun (2005) Current Opinion in
Plant Biology 8: 272-279
15
Genetically mappted TT genes involved in proanthocyanidin biosynthesis
(Zhao et al. (2010) Plant Physiology 153: 437)
tt mutants and their encoding
enzymes are as follows: tt4,
chalcone
synthase;
tt5,
chalcone
isomerase;
tt6,
flavanone 3-hydroxylase; tt7,
flavonoid 3′ hydroxylase; tt3,
dihydroflavonol 4-reductase;
ban, ANR; tt18 (at the same
locus as tt11), leucocyanidin
dioxygenase; tt10, laccase-like
polyphenol oxidase; tt12,
MATE antiporter; tt15, UDPGlc:sterol glycosyltransferase;
tt19 (at the same locus as
tt14), GST; aha10, P-type H+ATPase. tt mutants encoding
regulatory proteins are as
follows: tt1, WIK-type zinc
finger transcription factor; ttg1
(for transparent testa glabra1),
WD40 repeat transcription
factor;
tt2,
R2R3
Myb
transcription factor; tt8, basic
helix-loop-helix transcription
factor; tt16, MADS domain
transcription factor.
16
PA regulatory transcription factors (TT factors)
activate PA biosynthesis structural genes (TTs) in the
nuclei of seed coat endothelial cells under
appropriate conditions.
Proanthocyanidin transport and polymerization
Zhao et al. (2010) Plant Physiology 153: 437
PA pathway proteins are translocated to the
cytosolic side of the ER for synthesis of epicatechin
(white circles) and anthocyanins (red circles).
Epicatechin and anthocyanins are readily
glycosylated, and the conjugates are transported
into the vacuole by MATE (TT12) transporters. They
could also be loaded into the ER membrane system
or derived membrane vesicles, which are
transported to the central vacuole through
prevacuole compartment (PVC)-dependent vesicle
trafficking, or else they could be bound to the TT19
GST, which facilitates their transport into the ER,
vacuole, or other compartments.
The acidic vacuolar conditions may facilitate
nonenzymatic condensation of PA units, or the units
may undergo enzymatic condensation catalyzed by
TT10 or by yet unidentified proteins (red cylinders).
TT10 could be sorted and targeted within membrane
vesicles where is may catalyze the condensation of
PA units into oligomers. In the vacuole PA chain
elongation could be further catalyzed by TT10 using
epicatechin glucoside and PA oligomers as
substrates.
PAs can also be transported through membrane
vesicles or other mechanisms to the apoplastic
space, where they are subjected to oxidative
polymerization and further cross-linked with other
cell wall components, catalyzed by apoplastic TT10like polyphenol oxidases.
17
Phlobaphenes
http://www.biosci.ohio-state.edu/pcmb/osu_pcmb/faculty_sites/Erich/projects_phlobaphene.html
In maize, at least two flavonoid
biosynthetic
pathways
have
been
characterized
that
are
regulated
independently. One pathway results in 3hydroxy flavonoids such as anthocyanins
purple pigment, whereas the other
pathway produces 3-deoxy flavonoids such
as the phlobaphene red pigment
accumulated in kernel pericarp, silks and
cob.
Whereas the anthocyanin pathway and its
physiological functions in plant are well
documented, little is known about
phlobaphenes. One of the reasons is that
this family of flavonoids is not synthesized
in Arabidopsis thaliana, the model plant for
genetic and molecular biology. Moreover,
breeders
selected
maize
without
phlobaphenes for food industry (sweet
corn), because it alters the taste of the
kernels for consumers. Phlobaphene
biosynthesis results from the oxidation of
colorless
flavan-4-ol
monomers
or
polymers.
18
Isoflavonoids
(Dixon & Ferreira (2002) Phytochemistry 60, 205-211)
http://en.wikipedia.org/wiki/Image:To
fu-beijingchina.jpg
http://en.wikipedia.org/wiki/Image:
Lucerne_flowers.jpg
The Soy Health Claim
In October 1999, FDA approved a health claim that can be used on labels of soy-based
foods to tout their heart-healthy benefits. The agency reviewed research from 27
studies that showed soy protein's value in lowering levels of total cholesterol and lowdensity lipoprotein (LDL, or "bad" cholesterol).
Food marketers can now use the following claim, or a reasonable variation, on their
products: "Diets low in saturated fat and cholesterol that include 25 grams of soy
protein a day may reduce the risk of heart disease. One serving of (name of food)
provides __ grams of soy protein." To qualify for the claim foods must contain per
serving:
6.25 grams of soy protein
low fat (less than 3 grams)
low saturated fat (less than 1 gram)
low cholesterol (less than 20 milligrams)
sodium value of less than 480 milligrams for individual foods, less than 720 milligrams
if considered a main dish, and less than 960 milligrams if considered a meal.
19
Isoflavonoids - chemistry
(Veitch (2009) Natural Products Reports 26: 776)
20
Isoflavonoid biosynthesis
(Yu & Jez (2008) The Plant Journal 54: 750)
The flavonoid biosynthetic enzymes provide
naringenin (or liquiritigenin) for isoflavonoid
production (green).
An enzyme-catalyzed aryl migration reaction
generates the core isoflavonoid structure
(orange).
Additional modifications, indicated in blue,
lead to a variety of isoflavonoid compounds in
various plants (yellow).
21
Isoflavone synthase is a cytochrome P450-dep. monooxygenase catalyzing an aryl migration
(Steele et al. (1999) Archives of Biochemistry and Biophysics 367: 146)
Proposed mechanism of aryl migration by CYP93C2. Route a is the aryl migration of (2S)-flavanone to form the main
product, 2-hydroxyispflavanone. Route b is the hydroxylation of (2S)-flavanone at C-3 to form the by-product, 3hydroxyflavanone.
22
Complexes of HI4′OMT with
2,7,4′-Trihydroxyisoflavanone and
6aR,11aR-6a-Hydroxymaackiain.
(Liu et al. (2006) The Plant Cell 18: 3656)
(A) Stereo view of the electron density associated with the 2S,3Rstereoisomer of 2,7,4′-trihydroxyisoflavanone observed crystallographically
in the HI4′OMT complex.
(B) Stereo view of the electron density associated with 6aR,11aR-6ahydroxymaackiain observed crystallographically in the HI4′OMT complex.
(C) Close-up view of the HI4′OMT substrate/product binding site with SAH
and 2S,3R-2,7,4′-trihydroxyisoflavanone shown. The putative hydrogen
bonds are depicted as green spheres.
(D) Close-up view as in (C) illustrating the conformation and location of
bound 6aR,11aR-6a-hydroxymaackiain. For clarity, the active site residues
Asp-269, Phe-328, and Lys-337 that only participate in a hydrogen-bonding
network in the HI4′OMT-SAH-6a-hydroxymaackiain complex shown in (D)
were omitted in (C).
23
Glycosyltransferases of the flavonoid/isoflavonoid pathway can be promiscuous
(Wang (2010) Functional and Integrative Genomics 11: 13)
Isoflavonoids are often modified by
glycosylation with various sugars.
Crystal structures of five plant UGTs
have been determined.
Among these five UGTs, M. truncatula
UGT85H2
is
an
(iso)flavonoid
glycosyltransferase (Li et al. 2007); M.
truncatula UGT78G1 and grape (Vitis
vinifera) VvGT1 also recognize some
isoflavonoid compounds although they
are able to glycosylate cyanidin to yield
cyanidin 3-O-glycoside and may be
involved in the biosynthesis of
anthocyanins in vivo; M. truncatula
UGT71G1
also
recognizes
(iso)flavonoids in spite of its potential
role in saponin biosynthesis.
(a) Crystal structure of M. truncatula
UGT78G1 with bound UDP; (b) sugar
donor binding site and interaction
between the donor molecule UDPglucose and the enzyme; (c) acceptor
binding
pocket
with
UDP-2fluoroglucose (upper left) and acceptor
kaempferol (lower right).
24
Chemical structures of plant metabolites that
are commonly glycosylated
(Bowles et al. (2006) Annual Reviews in Plant
Biology 57: 567)
Glycosyltransferases of small molecules transfer sugars to a wide range of
acceptors, from hormones and secondary metabolites to biotic and abiotic
chemicals and toxins in the environment.
The enzymes are encoded by large multigene families and can be identified by
a signature motif in their primary sequence, which classifies them as a subset
of Family 1 glycosyltransferases.
The transfer of a sugar onto a lipophilic acceptor changes its chemical
properties, alters its bioactivity, and enables access to membrane transporter
systems.
In vitro studies have shown that a single gene product can glycosylate multiple
substrates of diverse origins; multiple enzymes can also glycosylate the same
substrate.
These features suggest that in a cellular context, substrate availability is a
determining factor in enzyme function, and redundancy depends on the
extent of coordinate gene regulation.
25