Abstract Arranging biological processes into ‘‘compartments’’ is a key feature of all eukaryotic cells. Through this mechanism, cells can drasti- cally increase metabolic efficiency and manage complex cellular processes more efficiently, sav- ing space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cyto- chrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabo- lons around P450s on the surface of the ER. We outline direct and indirect experimental data that describes P450 enzymes in the phenylpropa- noid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors. Keywords Cytochrome P450 Metabolon Enzyme interaction ER localization Cytochrome P450 reductase Abbreviations 4CL 4-Coumarate: CoA ligase AFM Atomic force microscopy C4H Cinnamate 4-hydroxylase CHI Chalcone isomerase CHS Chalcone synthase CPR NADPH-cytochrome P450 reductase DFR Dihydroflavonol reductase ER Endoplasmic reticulum F3H Flavanone 3b-hydroxylase F3¢H Flavonoid 3¢-hydroxylase F3¢5¢H Flavonoid 3¢,5¢-hydroxylase FLIM Fluorescence lifetime imaging microscopy FRET Fluorescence energy resonance transfer I2¢H Isoflavone 2¢-hydroxylase IFS Isoflavone synthase IOMT Isoflavone O-methyltransferase P450 Cytochrome P450 monooxygenase PAL Phenylalanine ammonia-lyase L. Ralston Sigma-Aldrich Biotechnology, Life Science and High Technology Center, 2909 Laclede Avenue, St. Louis, MO 63103, USA O. Yu (&) Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, Missouri 63132, USA e-mail: [email protected]Phytochem Rev (2006) 5:459–472 DOI 10.1007/s11101-006-9014-4 123 Metabolons involving plant cytochrome P450s Lyle Ralston Oliver Yu Received: 14 January 2006 / Accepted: 7 July 2006 / Published online: 28 October 2006 ȑ Springer Science+Business Media B.V. 2006
Transcript
Abstract Arranging biological processes into
‘‘compartments’’ is a key feature of all eukaryotic
esized to be facilitated by small accessory proteins
that mediate interactions between component
enzymes of a metabolon and cytoskeletal ele-
ments (Chuong et al. 2004; Graciet et al. 2004;
Winkel 2004). Since cellular processes must con-
stantly adjust in response to various environ-
mental and physiological stimuli, dynamic
formation and dissociation of metabolons may
provide an additional level of metabolic regula-
tion in biosynthetic pathways.
This review focuses on the organization of
metabolons around P450s in plant natural product
pathways. Beyond a metabolic role, P450s are
460 Phytochem Rev (2006) 5:459–472
123
also thought to play a structural role in these
complexes, providing a platform for their partners
to establish and maintain protein–protein inter-
actions. We will discuss the direct and indirect
evidence supporting the hypothesis of P450-an-
chored metabolons in several plant natural
product pathways.
Interactions between P450s and
NADPH-cytochrome P450 reductases
Given the central role of P450s in anchoring
metabolons of plant natural product biosynthesis,
the molecular architecture of the P450s provides a
framework for organizing larger macromolecular
complexes. All P450s rely on a heme co-factor to
split molecular oxygen, inserting one oxygen
atom to the substrate and reducing the other
oxygen atom to water. To accomplish this reac-
tion, a coupled, stepwise electron supply origi-
nating from NAD(P)H is indispensable (Denisov
et al. 2005). The electron supply is provided by
NADPH-cytochrome P450 reductase (CPR)
(Paine et al. 2005). Depending on the type of
P450, the CPR is either fused to the P450 to form
a multifunctional protein or operates as a sepa-
rate membrane-bound protein.
Structural and functional studies of the multi-
functional or ‘‘self-sufficient’’ P450s, such as the
fatty acid x-2 hydroxylase P450 BM3
(CYP102A1) from bacterium Bacillus megateri-
um, provide insight on the interaction between
the P450 and CPR. The multifunctional P450s
evolved through the fusion of the P450 and CPR
enzymes. This type of P450 consists of two dis-
crete domains that can be functionally separated
genetically and proteolytically. The first 472
amino-terminal residues of P450 BM3 comprise
the monooxygenase domain, while the remainder
of the enzyme makes up the reductase domain
(Munro et al. 2002). In P450 BM3, NADPH re-
duces a FAD flavin to a transient hydroquinone,
and a flavin mononucleotide (FMN)-binding do-
main shuttles electrons between the FAD in the
CPR domain and the heme in the P450 domain
(Sevrioukova et al. 1999). The fusion provides a
rare insight into the mechanism of the coupling of
the two units and how protein–protein interac-
tions facilitate the transfer of electrons (for re-
views see (Munro et al. 2002)). Structural and
biochemical data from P450 BM3 suggests that
the rate of electron transfer between NADPH
and FAD in the bipartate protein is much faster
than the observed catalytic activity of the isolated
monooxygenase domain (Munro et al. 1996). Two
basic residues, Lys572 and Lys580, stabilize the
hydroquinone-form of FAD and are essential
for efficient coupling of the electron transfer
chain (Sevrioukova et al. 1999; Murataliev and
Fig. 1 A plant cytochrome P450 localized to the corticalER membrane. (A) Soybean isoflavone synthase (IFS,CYP93C1) was fused in-frame with a yellow fluorescentprotein (EYFP) and transiently expressed in tobacco leafepidermal cells. IFS was localized to the ER network(yellow color, Ralston and Yu, unpublished data). (B) As a
control, soybean chalcone isomerase (CHI) was fused in-frame with a cyan fluorescent protein (ECFP) andtransiently expressed in tobacco leaf epidermal cells.Soluble CHI was localized to the cytoplasm and nucleus(green color). The size bar is 10 lm
Phytochem Rev (2006) 5:459–472 461
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Feyereisen 2000). Overall, for any P450 to be
functional, a tightly coupled CPR is essential.
In contrast to the multifunctional P450s, the
typical eukaryotic P450 interacts with a separate
membrane-bound CPR (Paine et al. 2005). The
amino-terminal region of CPR (approximately 60
amino acids in length) is a hydrophobic mem-
brane-spanning domain that anchors CPR to
the cytoplasmic surface of the ER. Similar to the
carboxyl-terminal region of the P450 BM3, the
function of the membrane-bound CPR is to ferry
electrons from NAD(P)H through FAD and
FMN to the P450 (Murataliev et al. 2004). Com-
pared to the vast number of P450 genes that exist
in plant genomes, the number of CPR genes is
much lower, and sequence homology is more
highly conserved than in P450s (Koopmann and
Hahlbrock 1997; Urban et al. 1997). This suggests
that a limited number of CPRs would couple with
a diverse array of P450 partners and, by exten-
sion, interact with a variety of metabolons.
Interactions between CPR and P450s have
been investigated mainly in mammalian systems.
A rat CPR without the amino-terminal mem-
brane domain has been crystallized (Wang et al.
1997). The enzyme is composed of three domains:
an FAD-binding domain similar to ferredoxin-
NADP+ reductase, an FMN-binding domain
similar to flavodoxins, and a linear linker that
connects the other two domains. The linker do-
main is responsible for the positioning of the co-
factors FMN and FAD in close proximity to the
heme of the P450 to facilitate efficient electron
transfer. Mutations in the linker region can cause
significant differences in the relative positions of
the two flavin domains and significantly reduce a
P450’s catalytic activities (Hubbard et al. 2001).
Similarly, removing the membrane anchor of a
mammalian CPR will produce a soluble and
enzymatically active CPR. However, the soluble
CPR will be unable to support the activity of a
P450 in vitro, due to the uncoupling of the elec-
tron transfer chain (Venkateswarlu et al. 1998).
The direct interaction of CPR with P450s was
visualized by atomic force microscopy (AFM) on
a reconstituted phospholipid bilayer disk con-
taining CPR and the P450 CYP2B4 (Bayburt
et al. 1998; Bayburt and Sligar 2002). CYP2B4
was found to protrude above the lipid surface,
3.5 nm. This is thought to orient the CPR such
that both the FMN- and FAD-binding domains
lie close to the membrane surface, allowing a
close interaction between FMN and the P450
heme (Wang et al. 1997). The lipid bilayer of the
ER membrane may also contribute to interac-
tions between CPR and P450 by altering the
composition of the bilayers to provide a relatively
stable support for the enzyme complexes when
needed (Ingelman-Sundberg et al. 1981, 1983).
More importantly, a group of basic residues,
Arg45, Lys46, Lys47, and Lys48, which lies at the
beginning of FMN-binding domain, may interact
with the phospholipid head groups of the mem-
brane and restrict the movement of CPR on the
membrane surface (Wang et al. 1997; Zhao et al.
1999). Similarly, a second group of basic residues
(Lys72, Lys74, Lys75, Arg78, Arg97, Lys100,
His103, and Arg108) at the surface of the FMN-
binding domain may provide an additional
membrane-binding site.
Surface residues on the P450 are also likely to
contribute to the CPR–P450 interaction. Specific
lysine and arginine residues on rat CYP1A1 are
shown to be involved in the formation of an
electron transfer complex with CPR (Shimizu
et al. 1991). Similarly, mammalian CYP2B1 and
CYP2B4 may also rely on complementary elec-
trostatic charges to bind with CPR (Shen and
Strobel 1993; Bridges et al. 1998). Site-directed
mutagenesis of CYP2B4 identified a series of ly-
sine and arginine residues on the proximal surface
of the heme region that interact with CPR’s FMN
region (Bridges et al. 1998). Moreover, chemical
cross-linking studies indicate that several acidic
residues on CPR are involved in the interaction
with CYP1A1 (Nadler and Strobel 1991). Other
evidence also suggests that the FMN-binding do-
main is the docking surface of the two enzymes,
as with P450 BM3. Electrostatic potential mea-
surement of the FMN-binding domain of a human
CPR shows clusters of acidic residues that could
form ion-pair interactions with a P450 (Shen and
Strobel 1995). In fact, a decreased association (Kd
10–90 nM) between rabbit CPR and CYP2B4 was
observed when the ionic strength of the buffer
was increased 10-fold (Davydov et al. 2000).
Mutations of the clusters located at the opposite
side of the FMN domain significantly reduces
462 Phytochem Rev (2006) 5:459–472
123
P450 activity, suggesting that the P450 may par-
tially ‘‘engulf’’ CPR at the FMN domain to bring
the heme and FMN in closer contact (Zhao et al.
1999).
In spite of the above evidence, the proposed
metabolon model has to reconcile with the fact
that, at least in mammalian cells, P450s are
present in a 10–25-fold molar excess over CPR
(Estabrook et al. 1971; Peterson et al. 1976). This
ratio dictates that any association of CPR with a
given P450 has to be transient and dynamic to
allow multiple P450s from different pathways to
have access to this essential partner. Such inter-
actions in plant cells are likely to be more com-
plicated due to the higher number of P450 genes
found in plant genomes.
Entry into the phenylpropanoid pathway:the CYP73A-related metabolon
Arguably the strongest physical evidence for the
existence of metabolic channels in a plant natural
product pathway involves the interaction between
the first two enzymes of the phenylpropanoid
pathway (Fig. 2A). The phenylpropanoid path-
way exists in all higher plants and is a major
consumer of energy and carbon fixed by photo-
synthesis (Werck-Reichhart and Feyereisen
2000). This pathway produces a myriad of
structurally diverse phenolic compounds, includ-
ing lignins, stilbenes, aurones, flavonoids, and
isoflavonoids. These compounds serve as signal
molecules, phytoalexins, anti-feedants, pigments,
UV protectants, and cell wall components in
plants. Since this pathway consumes large
amounts of biochemical resources, phenylpropa-
noid metabolism is tightly regulated both trans-
criptionally and post-transcriptionally (Weisshaar
and Jenkins 1998). Metabolic channeling between
enzymes at the entry point of pathway is sug-
gested to provide an additional level of regula-
tion.
The entry point enzyme of the phenylpropa-
noid pathway is phenylalanine ammonia-lyase
(PAL). PAL commits phenylalanine to the phe-
nylpropanoid pathway, deaminating phenylala-
nine and converting it into trans-cinnamate and
ammonia. In all species examined so far, PAL is
encoded by a multi-gene family (Dixon et al.
2002). Based on differential gene expression
patterns, it has long been suspected that the
Fig. 2 Schematic diagram of the phenylpropanoid (A)and dhurrin pathways (B). The P450 enzymes are in reditalics. Enzyme abbreviations are listed in the text, except
for CHR, chalcone reductase; STS, stilbene synthase; HID,2-hydroxyisoflavanone dehydratase; and IFR, isoflavonereductase
Phytochem Rev (2006) 5:459–472 463
123
various PAL isoforms are activated by different
environmental signals to divert phenylalanine
into different branches of the phenylpropanoid
pathway (Kao et al. 2002).
The second enzyme of the phenylpropanoid
pathway is the P450 cinnamate 4-hydroxylase
(CYP73A, C4H). C4H adds a hydroxyl group at
the C4 (para) position of trans-cinnamate to form
p-coumarate (Russell and Conn 1967; Schoch
et al. 2003). C4H is encoded by a single copy gene
in Arabidopsis and occurs in only one or two
copies in other species studied to date (Plant
Cytochrome P450 Database; http://www.drnel-
son.utmem.edu/P450dbplant.html). C4H was the
first plant P450 to be functionally characterized,
and it is among the most extensively studied of
the plant P450s (Chapple 1998).
PAL exists as an operationally soluble homo-
tetramer with no obvious membrane localization
domains (Wanner et al. 1995); however, a num-
ber of reports describe the association of PAL
with endomembranes in plant cells. In 1975,
based on sub-cellular fractionation and micro-
some enzyme activity assays, Czichi and Kindl
reported that PAL and C4H may assemble on
microsomal membranes as an enzyme complex
(Czichi and Kindl 1975). While C4H activity is
located exclusively in the microsomal fraction of
plant cells and PAL activity resides in the soluble
fraction of plant cell extracts, they noted that both
PAL and C4H activities co-localized to the
microsomal fraction. Substrate feeding experi-
ments suggested that cinnamate produced from
[3H]-phenylalanine by PAL is preferred over
exogenously added [14C]-cinnamate by C4H.
Hence, the majority of the C4H product,
p-coumarate, was derived from radiolabeled
phenylalanine (Czichi and Kindl 1977). Further
feeding assays carried out by others demonstrated
that disrupting the microsomal structure, which
may disrupt the interaction of the two enzymes,
led to the increased conversion of exogenously
added cinnamate conversion to p-coumarate
(Hrazdina and Wagner 1985). Taken together,
these earlier experiments provide strong indirect
evidence that PAL and C4H are co-localized on
the cytoplasmic surface of the ER and that cin-
namate is channeled directly to the active site of
C4H from PAL.
Rick Dixon’s lab has produced a good deal of
evidence for the existence of a tobacco PAL-C4H
metabolon. Rasmussen et al. demonstrated that
cinnamate produced from [3H]-phenylalanine
does not equilibrate with exogenously added
[14C]-cinnamate, indicating channeling between
PAL and C4H (Rasmussen and Dixon 1999).
Further, sub-cellular fractionation and protein gel
blot analysis demonstrated that tobacco PAL1 is
localized to both the soluble and microsomal
protein fractions, whereas the PAL2 isozyme is
strictly localized to the soluble fraction. More
recently, Achinine et al. provided more evidence
of direct enzyme interactions between PAL and
C4H. They used a combination of biochemical
and microscopic techniques to study the locali-
zation of PAL1, PAL2, and C4H (Achnine et al.
2004). Using these techniques, they verified the
sub-cellular localization of PAL1 and PAL2.
Moreover, they showed that over-expression of
C4H resulted in the ER localization of both
PAL1 and PAL2. This suggests that C4H itself
may be responsible for the membrane association
of PAL. Surprisingly, co-expression of unlabeled
PAL1 with PAL2-GFP resulted in a shift of flu-
orescence from ER membranes to the cytoplasm
in C4H over-expressing plants, whereas co-
expression of unlabeled PAL2 with PAL1-GFP
did not affect PAL1-GFP localization. This indi-
cates that PAL1 has a higher affinity for its
membrane localization site than PAL2. Fluores-
cence energy resonance transfer (FRET) studies
based on dual-labeling immuno-fluorescence
showed the PAL and C4H are located within
100 A of each other on the membrane. In addi-
tion, negative photo-bleaching results may sug-
gest that the connection between PAL and C4H is
fluid (Achnine et al. 2004).
In whole, there is extensive compelling evi-
dence suggesting that C4H anchors an ER-local-
ized metabolon at the entry point of the
phenylpropanoid pathway. However, at least one
example can be found in the literature disputing
the existence of a metabolon between PAL and
C4H. Ro and Douglas argue that enzyme kinetics
and biochemical coupling of PAL and C4H are
enough to drive carbon flux into the phenyl-
propanoid pathway (Ro and Douglas 2004). They
reconstituted the entry point of the pathway by
464 Phytochem Rev (2006) 5:459–472
123
expressing PAL, C4H, and CPR in yeast. They
then fed the engineered yeast [3H]-phenylalanine
and [14C]-cinnamate simultaneously. In this sys-
tem, inhibition of C4H by P450 inhibitors indeed
reduced PAL activity, demonstrating biochemical
coupling. However, unlike experiments using
plant microsome fractions, the two radiolabeled
substrates showed equal accessibility to C4H,
indicating that no channeling occurs between
PAL and C4H. Earlier analysis using transgenic
plants supported this biochemical coupling the-
ory: when the C4H gene was silenced in tobacco,
PAL activity was significantly reduced while PAL
silencing did not disturb C4H activity. This data
provides further evidence of a feedback loop at
the entry point of the phenylpropanoid pathway
(Blount et al. 2000). Clearly, biochemical cou-
pling is part of the regulation mechanism between
PAL and C4H. Yet the physical interactions be-
tween these enzymes inside plant cells should not
be excluded based on experiments in yeast.
Cytoskeletal elements and accessory proteins
critical for interactions between PAL and C4H
may well be lacking in a heterologous host. It is
possible that at the entry point of the phenyl-
propanoid pathway, both regulation mechanisms
co-exist inside plant cells.
Flavonoid biosynthesis: CYP75B-related
metabolon
While the majority of the carbon flux into the
phenylpropanoid pathway is directed towards
lignin production (discussed below), the second
greatest carbon sink from this pathway is flavo-
noid biosynthesis. Flavonoid compounds are
produced by the coordinate action of a series of
operationally soluble and membrane-bound
cytoplasmic enzymes (Fig. 2A) (Winkel-Shirley
2001a). Soluble enzymes of the flavonoid branch
pathway include 4-coumarate: CoA ligase (4CL),
chalcone synthase (CHS), chalcone isomerase
(CHI), flavanone 3b-hydroxylase (F3H), and di-
hydroflavonol reductase (DFR). A number of
P450s also participate in this branch of the path-
way. Flavonoid 3¢-hydroxylase (CYP75B, F3¢H)
modifies the B-ring of the flavanone backbone at
the C3¢ position. Another P450, flavonoid 3¢,5¢-
hydroxylase (CYP75A, F3¢5¢H), is found in a
limited number of species and modifies the B-ring
at the C5¢ position (Winkel-Shirley 2001b).
There is strong evidence suggesting that the
operationally soluble enzymes of the flavonoid
branch pathway participate in metabolons. In a
yeast two-hybrid assay, Burbulis and Winkel-
Shirley demonstrated that CHS, CHI, and DFR
interact with one another in an orientation-
dependent manner (Burbulis and Winkel-Shirley
1999). Affinity chromatography and immuno-
precipitation assays also demonstrated interac-
tions between CHS, CHI, and F3H in lysates from
Arabidopsis seedlings (Burbulis and Winkel-
Shirley 1999). Although three-dimensional struc-
tures are available for CHS and CHI (Ferrer et al.
1999; Jez et al. 2000), no structural model of this
proposed macromolecular complex has been
published. However, mechanistic studies of CHS
and CHI suggest the need for metabolic chan-
neling in this pathway. The non-enzymatic cycli-
zation of chalcones into flavanones readily occurs
in solution but yields an enantiomeric mix of
biologically inactive and active isomers. Chan-
neling between CHS and CHI would prevent the
formation of mixed isomers (Jez et al. 2002).
Interestingly, the catalytic efficiency of CHI (kcat/
Km = 109 M–1 min–1) approaches the diffusion
limit, so one might ask why CHI would need
metabolites to be directed towards it. One possi-
ble answer is that the moderately lipophilic nat-
ure of chalcones and flavanones may require
channeling to limit the potential for sequestration
in cellular membranes. Moreover, in legumes,
channeling through the metabolon may ‘‘slow’’
the activity of CHI and protect the chalcone pool
from complete conversion into naringenin (Jez
et al. 2002).
There is no direct evidence that the complex
formed by CHS, CHI, F3H, and DFR interacts
with P450s. However, if these enzyme do form
such an interaction, the most likely candidate is
F3¢H (or perhaps F3¢5¢H, if it is expressed). The
complex could also involve the upstream P450
C4H, thus forming a metabolon that encompasses
the majority of the enzymes of the general phe-
nylpropanoid and flavonoid branch pathways
(Winkel 2004; Jorgensen et al. 2005). There is
interesting indirect evidence suggesting that CHS/
Phytochem Rev (2006) 5:459–472 465
123
CHI-related metabolons are indeed localized to
the ER. First, CHI contains a motif that indicates
the enzyme might be modified post-translation-
ally by a fatty acid moiety with a thiol-sensitive
linkage. This would provide a mechanism for the
membrane localization of this soluble enzyme
(Burbulis and Winkel-Shirley 1999). Earlier re-
ports indicate that in the hypocotyl epidermis of
buckwheat (Fagopyrum esculentum), CHS activ-
ity and protein were detected at the cytoplasmic
face of the rough ER, based on linear sucrose
density gradient fractionation, immuno-blots, and
immuno-gold analysis (Hrazdina et al. 1987). Fi-
nally, membrane localization of CHS, and co-
localization of CHS and CHI were observed at
the ER and tonoplast in Arabidopsis root cells
(Saslowsky and Winkel-Shirley 2001). The anti-
bodies detected an electron-dense region with
membrane structures. In the Arabidopsis tt7 mu-
tant in which the P450 F3¢H is deleted, the elec-
tron-dense regions containing these two enzymes
were not detected. However, CHS and CHI were
still found to co-localize to the ER and tonoplast
in the tt7 mutant, suggesting that other proteins
may function in recruiting the soluble flavonoid
pathway enzymes to membranes (Saslowsky and
Winkel-Shirley 2001). Interestingly, recent data
suggests that flavonoids and CHS and CHI pro-
teins are also localized to nucleus of Arabidopsis
(Saslowsky et al. 2005). However, the implica-
tions of this discovery are not yet clear. Collec-
Medicago family, the first such modification is the
methylation of the 4¢-hydroxy on the B-ring of
the isoflavones. This step is mediated by an
operationally soluble isoflavone-O-methyltrans-
ferase (IOMT).
Liu et al. observed that IOMT from alfalfa
(Medicago sativa) was translocated to the ER upon
elicitor treatment, co-localizing with IFS (Liu and
Dixon 2001). They also noted that feeding studies
in alfalfa cell suspension cultures suggested chan-
neling between IFS and the IOMT. Radiolabel
from [3H]-liquiritigenin was preferentially incor-
porated into formononetin, the product of IOMT,
as well as other downstream isoflavonoids. How-
ever, radiolabel from daidzein, the in vitro sub-
strate of IOMT, was not incorporated into
downstream isoflavonoids. This data suggests that
the methyltransferase is closely associated with
IFS, facilitating rapid methylation and stabilization
of the product of IFS. This association was also
used to explain the observed differences in IOMT
regio-specificity between in vivo and in vitro assays
(Liu and Dixon 2001). However, different types of
IOMTs have been cloned, indicating that these
differences in regio-specificity may instead be
caused by the activity of different IOMT enzymes
(Akashi et al. 2000, 2003). Liu et al. provided a
model suggesting that intermediates of the isofl-
avonoid pathway could rapidly flow from one en-
zyme center anchored by IFS to next enzyme
center in a metabolon anchored by another P450,
isoflavone 2¢-hydroxylase (CYP81E, I2¢H), and
eventually enter into the vacuole (Liu and Dixon
2001). This process is more complicated in other
legume species. In these species, the product of
I2¢H is prenylated by plastid-associated pren-
yltransferases to form pterocarpans and fur-
anocoumarins before entering the vacuole (Dhillon
and Brown 1976). This must require the shuttling of
compounds between different organelles for the
metabolon model to hold true.
The association of IFS with upstream enzymes
such as CHI and CHS can only be implied. Sev-
eral groups have shown that efforts to engineer
isoflavonoid synthesis in non-legume plants by
466 Phytochem Rev (2006) 5:459–472
123
introducing IFS resulted in a disproportionate
decrease in flavonoid production, which cannot
be explained by a shift in metabolic flux alone
(Yu et al. 2000; Liu et al. 2002). In transgenic
Arabidopsis expressing IFS, the production of
genistein can be increased 3-fold by irradiation
with UV light, but the ratio of genistein in total
flavonoid levels actually decreased. This suggests
that the pool of naringenin substrate is not
equally accessible to the flavonoid and isoflavo-
noid branches of the pathway (Yu et al. 2000).
Similar experiments using transgenic tobacco and
maize cell cultures demonstrated similar ‘‘com-
partmentalization’’ of the naringenin substrate
(Yu et al. 2000). Additionally, the treatment of
tobacco leaves with UV light to increase na-
ringenin levels resulted in elevated flavonol levels
but failed to raise anthocyanin or isoflavone lev-
els. This data suggests that flux through the phe-
nylpropanoid pathway is tightly channeled to
flavonol production in tobacco leaves, and IFS
has difficulty accessing this substrate.
To delineate enzyme interactions between IFS
and endogenous flavonoid pathway genes, a CHI
from alfalfa, shown to be an legume-specific
isomerase by its ability to convert isoliquiritigenin
into liquiritigenin, was transformed into Arabid-
opsis and then genetically crossed into a trans-
genic Arabidopsis line carrying the IFS gene (Liu
et al. 2002). The combination of genes enhanced
genistein accumulation moderately. However,
flavonol accumulation was significantly reduced
compared to transgenic plants carrying only the
legume-specific CHI. The disproportionate
reduction of flavonol biosynthesis caused by the
presence of IFS further confirmed that the flux of
substrate is preferentially channeled towards
endogenous flavonoid biosynthesis (Liu et al.
2002). One possible explanation offered for this
outcome is that the membrane-localized IFS
might interfere with the assembly of metabolic
complexes dedicated to flavonoid biosynthesis,
which may be anchored to the ER by the P450
F3¢H in Arabidopsis (Yu et al. 2000). When the
IFS and CHI were introduced into a tt6/tt3 double
mutant background, genistein accumulation was
enhanced by up to 30-fold as compared to plants
expressing IFS alone. This mutant has structural
defects in both F3¢H and DFR genes and is thus
blocked in flavonol and anthocyanin production.
Again, this suggests that the bottleneck for isof-
lavone production in Arabidopsis is competition
for flavanones between IFS and endogenous fla-
vonol biosynthetic enzymes (Liu et al. 2002).
This brings us to an interesting topic regarding
the regulation of the competing branch pathways.
In legumes, where the flavonoid and isoflavonoid
pathways co-exist, the flux of flavanone interme-
diates should be differentially divided into the
two pathways in response to abiotic and biotic
stresses. Plants can activate the expression of a
subset of genes that divert the precursor phenyl-
alanine into specific products of the pathway
using a number of relatively well-defined tran-
scription factors (Yu et al. 2003). However, me-
tabolons could play a major role in controlling
flux through the pathway. In fact, two functionally
different CHI exist in legumes (Shimada et al.
2003; Ralston et al. 2005): the type I CHI exists in
all higher plants and converts tretrahydroxy-
chalcones into the flavanone naringenin, while the
legume specific type II CHI converts both tetra-
hydroxy-chalcones and trihydroxy-chalcones into
naringenin and liquiritigenin, respectively. The
two types of CHI are differentially expressed, and
only the type II CHI is induced by defense signals
(Ralston et al. 2005). It’s possible that the type II
CHI, together with other isoflavone biosynthetic
enzymes may form their own metabolic channels,
independent of the flavonoid channels described
in Arabidopsis (Fig. 3) (Winkel 2004; Yu and
McGonigle 2005).
Cyanogenic glucoside biosynthesis: CYP79A/
CYP71E-related metabolons and others
The biosynthesis of cyanogenic glucosides in sor-
ghum adds another dimension to the involvement
of P450s in metabolons. Dhurrin is a type of cya-
nogenic glucoside that is produced from the amino
acid tyrosine by two P450s, CYP79A1 and
CYP71E1, and the UDP-glucosyltransferase
UGT85B1 (Fig. 2B) (Celenza 2001; Morant et al.
2003; Jorgensen et al. 2005). Feeding assays using
the radioisotopically labeled substrate tyrosine and
the intermediate Z-p-hydroxyphenylacetal dox-
ime demonstrated clearly that the two P450s are
Phytochem Rev (2006) 5:459–472 467
123
highly channeled, with the majority of the radio-
label in dhurrin coming from tyrosine (Moller and
Conn 1980). Epitope tagging and confocal laser
scanning microscopy revealed that this metabolon
is located in a distinct domain of the ER (Nielsen
and Moller 2005). From an evolutionary point of
view, the coupling of the three enzymes likely arose
out of the need to prevent the highly labile and
toxic p-hydroxymandelonitrile intermediate from
releasing cyanide into the cytoplasm.
Genetic engineering has provided further sup-
port for the existence of P450 anchored metabo-
lons. When the entire cyanogenic glucoside
pathway was introduced into Arabidopsis by
expressing all three enzymes, the production of
dhurrin had virtually no effect on the metabolic
profiles of the host plants (Kristensen et al. 2005).
This toxic product was effectively contained and
sequestered in vacuoles. However, when only the
two P450s were expressed in Arabidopsis, without
the metabolon partner UGT85B1, plants showed
a variety of toxicity-related phenotypes. Inter-
estingly, when introduced separately into Ara-
bidopsis, the first enzyme CYP79A1 was able to
establish highly efficient interactions with down-
stream glucosinolate-producing enzymes, and al-
tered the overall glucosinolate profile of
Arabidopsis (Bak et al. 1999).
With the rapid advances in the understanding
of cellular and biochemical processes, metabolons
have been invoked in other metabolic pathways.
For example, the lignin biosynthetic enzymes
have previously been proposed to form a meta-
bolic grid, in which the flux could proceed in a
relatively random manner (Humphreys and
Chapple 2002; Boudet et al. 2003), however, re-
cent molecular analysis, particularly the new en-
zyme kinetic and substrate specificity
observations suggested that many of the routes
through the initially proposed grid are unlikely to
function in vivo, thus suggesting a more or less
linear metabolic pathway. Dixon et al. proposed
an interesting model suggesting that the linearity
of enzyme reactions is the result of metabolic
channeling among the key lignin enzymes (Dixon
et al. 2001). The metabolons are thought to pre-
vent the intermediates from coming into contact
with competing enzymes that might lead to the
development of a less efficient metabolic grid.
However, direct evidence of enzyme interactions
in this pathway is scarce. While the existence of
metabolons has been postulated in other plant
natural product pathways, including the alkaloid
and terpenoid pathways (Chappell 1995; Meme-
link 2004), investigators have yet to produce
compelling evidence of this phemonenon.
Conclusion and perspectives
A considerable amount of evidence argues for the
formation of metabolic P450-anchored enzyme
complexes in plant natural product pathways.
However, unlike the more conventional concept
of the stable multienzyme complexes that are
focused on completing one particular task more
efficiently, the P450-related metabolons probably
take on broader roles in regulating complicated
pathways and even metabolic grids. Such func-
tions require these metabolons to be able to
accommodate the constantly changing environ-
ment both inside and outside of the cell. There-
fore, most of these metabolons are likely to be
very dynamic and transient. The assembly and
Fig. 3 Proposed models of the flavonoid (A) and isofl-avonoid (B) metabolons. The enzyme abbreviations arethe same as in Fig. 2, except for UFGT, UDP-glucose:anthocyanidin-3-O-glucosyl trasnsferase; LDOX, leuco-anthocyanidin deoxygenase
468 Phytochem Rev (2006) 5:459–472
123
disassociation of enzymes within these complexes
is expected to occur rapidly in response to inter-
nal and external stimuli. As a consequence, it has
been very difficult to isolate and study these
complexes. Traditional biochemical assays,
including isotope dilution, substrate feeding, and
sub-cellular fractionation, while valuable, are
limited in their ability to provide direct evidence
of enzyme interactions. Co-immunoprecipitation,
protein cross-linking and affinity purifications are
best suited for stable interactions (Blancaflor and
Gilroy 2000); and yeast two-hybrid assays are not
applicable for membrane bound P450s. There-
fore, newer technologies, such as multiphoton
confocal co-localization (Blancaflor and Gilroy
2000), FRET and fluorescence lifetime imaging
microscopy (FLIM) analysis (Wallrabe and Pe-
riasamy 2005), split ubiquitin assays (Thaminy
et al. 2004), protein fragment complementation
assays (Subramaniam et al. 2001), and surface
plasmon resonance assays (Magee et al. 2002)
may provide direct and in vivo enzyme interac-
tion analysis.
If one or more plant P450 structures can be
resolved by X-ray crystallography, it will certainly
provide tremendous help in understanding of
nature of these protein–protein interactions, since
many of the soluble partners of P450s have al-
ready been resolved structurally. This will allow
directed mutagenesis to be carried out to evaluate
the interactions at the interface of these proteins.
However, the crystallization of membrane-bound
proteins is notoriously difficult and may require
intensive labor and extensive modifications of the
P450. In addition, direct visualization based on
AFM (Janovjak et al. 2005) or cryo-EM (Chiu
et al. 2005) is also possible if the enzyme com-
plexes can be functionally reconstituted or iso-
lated.
Understanding the structure and function of
the P450-related metabolons clearly is a consid-
erable challenge. However, these complexes very
likely provide another layer of cellular regulation
mechanisms and play important roles in plant
growth and development. The information ob-
tained on metabolons will also guide new meta-
bolic engineering efforts and allow the plant
community to harvest the desired products with
more certainty and higher efficiency.
Acknowledgements We would like to thank Dr. Joe Jezof the Danforth Center for the critical review of thismanuscript and for many thoughtful discussions on thetopic. The research in Dr. Yu’s lab is supported by theNational Science Foundation (MCB0519634), UnitedStates Department of Agriculture (CSREES: 2005-05190),and Missouri Soybean Merchandising Council (02-242).
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