Download Cytochrome P450-enzymes involved in the biosynthesis of mono

Phytochem Rev (2015) 14:7–24
DOI 10.1007/s11101-013-9280-x
Cytochrome P450-enzymes involved in the biosynthesis
of mono- and sesquiterpenes
Corinna Weitzel • Henrik Toft Simonsen
Received: 29 August 2012 / Accepted: 5 March 2013 / Published online: 17 March 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Terpenoids form the largest group of plant
specialized metabolites and exhibit essential functions
in plant metabolism, propagation and defence. Since
several mono- and sesquiterpenoids, like artemisinin,
menthol and nootkatone, have proven beneficial for
mankind, they also possess high socio-economic
value. The general mechanisms of terpene biosynthesis are understood and enzymes catalysing the formation of the isoprenoid basic carbon skeletons have
been described frequently. In the subsequent pathway
steps, it is mainly cytochromes P450 that catalyse the
decoration of these basic skeletons and thereby
contribute significantly to the structural diversity
observed. Structure–function relationship, even
though discussed intensively, is poorly understood
for this enzyme family; even with the phylogenic
relationship well established identification of the
functionality of the single enzymes is challenging,
and, so far, only a few have been characterized. This
review provides an overview over cytochromes P450
participating in the biosynthesis of mono- and sesquiterpenes. Only enzymes that have been described
thoroughly after purification and heterologous expression are included in this review and their characteristic
features are discussed.
C. Weitzel H. T. Simonsen (&)
Department of Plant and Environmental Sciences, Faculty
of Science, University of Copenhagen, Thorvaldsensvej
40, 1871 Frederiksberg C, Denmark
e-mail: [email protected]
Keywords Cytochrome P450 Isoprenoids Plant
specialized metabolism Terpenoids
Abbreviations
CPR Cytochrome P450 reductase
CYP Cytochrome P450
GAO Germacrene acid oxidase
SRS
Substrate recognition site
TPS
Terpene synthase
Introduction
Plant cells are capable of forming an overwhelming
variety of specialized metabolites, both in terms of
complexity and quantity. These small organic molecules allow plants to cope with various types of stress,
and often have biological activities beneficial to
humans that make them of high commercial interest
to the pharmaceutical and biotechnological industry
(Cragg et al. 2011). Many of these biologically active
compounds are terpenoids.
With several tens of thousands known structures,
terpenoids form the largest class of plant natural
products (Buckingham 2011). Some terpenoids are
key constituents for cellular functions (e.g., electron
transport chains, steroidal membrane components, and
hormones), but many of them are specialized metabolites that influence the fitness of the synthesizing
organism (Ashour et al. 2010). Plant terpenoids are
known to serve as insect and herbivore deterrents,
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allochemicals, and as attractants for pollinators and
beneficial insects in tritrophic interactions (Gershenzon and Dudareva 2007). As a result, a universal
statement about spatial and temporal patterns of
accumulation is not possible (Gershenzon et al.
2000). In some plants, terpenoids are synthesized in
specialized physical structures such as oil glands or
resin ducts (Miller et al. 2005), whereas in others
biosynthesis is not even restricted to particular organs
but proceeds basically in all tissues (Bohlmann and
Keeling 2008). Since many terpenoids serve as
defence compounds, their biosynthesis is often
induced by stress, and regulation seems mainly to
take place at transcriptional level (Crocoll 2011).
Besides their eco-physiological role, terpenoids
have also benefited mankind as flavours, fragrances,
pharmaceuticals, nutraceuticals, and industrial chemicals (Berger 2007; Zwenger and Basu 2008). Biotechnological production is of special interest for
terpenoids with known pharmacological properties or
with application in foods and fragrances, since their
limited availability in nature and the structural complexity of the molecules can make it the only
commercial sustainable method of production (Daviet
and Schalk 2010). This however requires profound
knowledge on the presence of terpenoids in plants and
their biosynthesis (Simonsen et al. 2009).
The wide range of physiological activities and
industrial applications exhibited by terpenoids can be
attributed to their structural diversity that is a result of
the catalytic plasticity of terpene synthases (TPSs).
TPSs convert simple linear prenyldiphosphates into
complex terpenes, often with cyclic groups and a large
number of chiral centres (Bohlmann et al. 1998).
Following the core terpenoid formation further modifications are often catalysed by cytochromes P450
often leading to oxidations of the terpenoid. Additionally, also dehydrogenases and acyltransferases
have been shown to contribute to the structural
diversity of terpenes (Ashour et al. 2010), furthermore
methylation, acylation, and glycosylation of terpenoids are described (Mau and Croteau 2006). In this
review, we aim to provide an overview of cytochromes
P450 involved in mono- and sesquiterpene biosynthesis. The focus is only on those enzymes that have been
characterized through cloning and expression. Examinations where only whole cell extracts, microsomes
or likewise have been studied are not included due to
the complexity of these system, thus leading to results
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Phytochem Rev (2015) 14:7–24
of less clarity. Along with in vitro and heterologous
characterization, in planta characterization should be
pursued for all cytochromes P450.
Cytochromes P450 form one of the largest and
oldest gene superfamilies and can be found in the
genome of all biological kingdoms (Nelson 2011). The
result of a cytochrome P450 catalysed reaction is most
often insertion of oxygen (hydroxylation), although
also dehydrogenation, isomerization, dimerization,
carbon–carbon bond cleavage, reductions, as well as
N-, O- and S-dealkylations, sulphoxidations, epoxidations, deaminations, desulphurations and several
more have been described (Bernhardt 2006; Guengerich 2001; Sono et al. 1996). The term ‘P450’ is not
based on a common enzymatic function, but their
shared ability to display a typical absorption band at
450 nm when carbon monoxide is bound to the
reduced form of this b-type heme (Omura and Sato
1964). Based on their amino acid sequence, cytochromes P450 are classified into families (minimum
40 % sequence identity) and subfamilies (greater than
55 % sequence identity) (Werck-Reichhart and Feyereisen 2000). Until summer 2012, a nomenclature
committee led by Prof David Nelson, The University
of Tennessee Health Science Center, named over
18,000 genes attributed to several hundred families
(Nelson 2012).
Generally, a common overall topology and
tridimensional fold of all cytochromes P450 have
been observed (Graham and Peterson 1999) despite
their relative low sequence similarity on amino acid
level, which can be less than 20 % even within a
plant species (Bak et al. 2011). Only a few amino
acids are strictly conserved. These are found in the
core where they form a four-helix (D, E, I and L)
bundle, two sets of b-sheets, and a coil, generally
known as the ‘meander’. The catalytic centre
consists of a central heme-group bound to the
thiolate of a highly conserved cysteine residue
(Hasemann et al. 1995; Peterson and Graham 1998).
This structural conservation allows for a common
reaction mechanism of electron and proton transfer
followed by oxygen activation, which has been
described in detail by Hamdane et al. (2008). In
contrast, little is known about substrate specificity
and type of reaction catalysed by the individual
enzymes, since these are controlled by less conserved regions of the protein (Werck-Reichhart and
Feyereisen 2000).
Phytochem Rev (2015) 14:7–24
Determination of candidate genes and investigation
of enzyme features
Within plants, cytochromes P450 take part in numerous processes of general and specialized metabolism
and thus play important roles in the biosynthesis of
plant hormones, in cell wall biosynthesis as well as in
the biosynthesis of specialized compounds involved in
defence or for the attraction of pollinators, to mention
just a few examples (Mizutani 2012). Hence, they are
abundantly present in genomes and transcriptomes of
all plants. So far, 127 plant cytochrome P450-families
have been described. The latest plant cytochrome
P450-family was added in 2005, implying that the
diversification of plant cytochromes P450 seems to be
widely covered; the appearance of new subfamilies,
however, still continues (Nelson and Werck-Reichhart
2011).
The discovery of cytochromes P450 catalysing
hydroxylation steps in the biosynthesis of mono- and
sesquiterpenes is impeded by many factors that
concern determination of the right candidate gene
and investigation of the enzyme’s features. Structure–
function relationship of plant cytochromes P450 is still
poorly understood, and, for this reason, has not lead to
substrate discovery. On the other hand, phylogeny
could prove a promising approach to limit the possible
enzyme functions. Even though enzymatic function of
many cytochromes P450 has not been revealed, it has
generally been recognized that cytochromes P450 in
the same family or subfamily catalyze similar reactions or are involved in the same biosynthetic pathway
(Nelson and Werck-Reichhart 2011). Until now,
cytochromes P450 contributing to the metabolism of
specialized terpenoids are described in 4 plant clans
(Hamberger and Bak 2013). This finding indicates that
the functions of CYP clans and families are more often
a result of evolution than of sequence relatedness
(Nelson and Werck-Reichhart 2011), and especially
within the CYP71 family, species-specific rather than
conserved functions exist (Devi et al. 2011). So far,
most terpenoid related cytochromes P450 are members of the CYP71 clan (Fig. 1), a huge clan that
comprises cytochromes P450 involved in the metabolism of the majority of specialized compounds
(Hamberger and Bak 2013). First, it has mainly been
genes from the CYP71A and CYP71D subfamilies
that have been associated with oxidation of mono- or
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sesquiterpenes in different plant species of various
families (Hallahan et al. 1994; Mau and Croteau 2006;
Ralston et al. 2001; Takahashi et al. 2007), but lately,
many new CYP71-subfamilies have been described,
many representing enzymes catalysing oxygenation of
small molecules, also sesquiterpenoids (Nelson and
Werck-Reichhart 2011). Exceptional is CYP72A1 that
catalyses formation of secologanin, a monoterpenoid
and precursor of the indole alkaloid strictosidine
(Irmler et al. 2000; Yamamoto et al. 2000). Members
of the same subfamily, CYP72A, take part in triterpene biosynthesis, other CYP72-families are responsible for the catabolism of di- and triterpenoid
phytohormones (reviewed by Hamberger and Bak
2013).
Discovery of pathway specific cytochromes P450
and their involvement in terpenoid biosynthesis
require thorough biochemical characterization. Generally, high substrate specificity of the enzymes has
been noticed (Schuler and Werck-Reichhart 2003),
hampering this task exceedingly. Accessibility to a
certain substrate can be difficult, because many are not
commercially available. Several simple monoterpenoids can by chemical synthesis. But for many highly
decorated monoterpenoids and most sesquiterpenoids,
chemical synthesis can be laborious, inefficient and
costly, if possible at all, due to the complex chemical
structures. Co-expression of the candidate genes with
genes upstream in the proposed pathway can circumvent this problem and is performed more and more
often (Greenhagen et al. 2003; Ikezawa et al. 2011);
efficient expression of all genes and transcription into
functional enzymes in vitro must be ensured and indepth biochemical characterization of all novel
enzymes is needed.
Recently, various cytochromes P450 capable of
accomplishing successive hydroxylation steps were
described. These enzymes do not only introduce a
hydroxyl group, but further catalyse its subsequent
oxidation into a ketone or carbonyl group; some even
accept various terpenes, alcohols and aldehydes as
substrates. This is still a subject that needs further
attention, but it seems to indicate the possibilities for
broader substrate acceptance and a wider product
portfolio. With this review we aim to establish a
current overview and enable further investigation into
terpenoid biosynthesis and how cytochromes P450
play a role in this.
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Fig. 1 A phylogenetic tree showing the cytochromes P450 discussed in this review
Cytochromes P450 involved in monoterpene
biosynthesis
Monoterpenoids have been detected in more than 2000
plant species throughout the plant kingdom and, so far,
more than 3,400 different monoterpenoids have been
described (Buckingham 2011). The arrangement of
the carbon skeleton can be acyclic, mono- and
bicyclic. Tricyclic monoterpenoids exist, but they
are rare. Monoterpenoids are the major components of
most plant essential oils (Evans 2009). In many plant
species, essential oil is produced and stored in
specialized cells, glandular trichomes, oil glands etc.
(Evans 2009). In some cases, these cells could be
isolated and served as an enriched source of biosynthetic enzymes and their corresponding transcripts
(Mau and Croteau 2006).
In 1992, the function of the first plant cytochrome
P450, CYP71A1, was described (see Table 1). The
research group headed by Dr. Wallsgrove had isolated
it from the mesocarp of ripe avocado fruits (Persea
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americana, Lauraceae) and demonstrated its ability of
hydroxylation of the monoterpenoids nerol and geraniol. Surprisingly, neither monoterpenoids nor derived
products have been found in the original tissue
(Hallahan et al. 1992). Subsequently, various studies
have shown that CYP71A1 is also capable of binding a
number of substrates; for instance, it carries out a
monooxygenation reaction on p-chloro-N-methylaniline (pCMA). The function of the enzyme in vivo,
however, remains unclear (Hallahan et al. 1994).
Southern blot analysis revealed the existence of a
closely related cytochrome P450 expressed in catmint
(Nepeta racemosa, Lamiaceae) leaves, this enzyme also
catalyses the hydroxylation of nerol and geraniol. The
catmint cytochrome P450 specifically hydroxylates
both monoterpenes at C10, while CYP71A1 was shown
to catalyse 2,3- or 6,7-epoxidation (Hallahan et al.
1994). CYP71A5 and CYP71A6 were eventually
cloned from N. racemosa leaves. Participation of
CYP71A5 in monoterpene biosynthesis, probably as
geraniol 10-hydroxylase, was deduced from expression
71A32
71A6
71A5
71A1related
Persea
americana
71A1
(Lamiaceae)
Mentha x
piperita
(Lamiaceae)
Nepeta
racemosa
(Lamiaceae)
Nepeta
racemosa
(Lauraceae)
Plant origin
Cytochrome
P450
OH
OH
OH
OH
OH
nerol
10-hydroxy-nerol
geraniol
10-hydroxy-geraniol
Proposed function: geraniol-10-hydroxylase; no functional characterization after heterologous expression
OH
Catalysed reaction
Table 1 Cytochrome P450 genes involved in monoterpene biosynthesis
AF346833
Y09424
Y09423
M32885
Gen Bank
number
Bertea et al. (2001), Mizutani and Sato
(2011)
Clark et al. (1997)
Hallahan et al. (1994)
Hallahan et al. (1992, 1994)
References
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123
123
Fragaria vesca
71AR1
Mentha spicata
71D18
71D174
(Lamiaceae)
71D15
(Lamiaceae)
Perilla frutescens
(Lamiaceae)
Mentha x piperita
71D13
(Rosaceae)
Plant origin
Cytochrome
P450
Table 1 continued
Catalysed reaction
GQ120438
AF124815
AF124817
AF124816
Sequence published in reference only
Gen Bank number
Mau et al. (2010)
Haudenschild et al. (2000), Lupien et al. (1999)
Haudenschild et al. (2000), Lupien et al. (1999)
Aharoni et al. (2004)
References
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72A1
Thymus
vulgaris
71D178182
(Apocynaceae)
Catharanthus
roseus
(Lamiaceae)
Origanum
vulgare
Plant origin
Cytochrome
P450
Table 1 continued
HO
OH
O
O
HO
In addition, a-terpinene, (?)-R-limonene, (-)-S-limonene and (-)-R-a-phellandrene are converted
-terpinene
Catalysed reaction
thymol
carvacrol
OH
Irmler et al. (2000), Yamamoto
et al. (2000)
Crocoll (2011)
Sequences awaiting
publication
AAA33106
References
Gen Bank number
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13
123
Collu et al. (2001)
ACZ48680
CAC80883
(Gentianaceae)
Swertia mussotii
76B10
Catharanthus roseus
76B6
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(Apocynaceae)
Plant origin
Cytochrome
P450
Table 1 continued
Catalysed reaction
Gen Bank number
Wang and Essenberg (2010)
Phytochem Rev (2015) 14:7–24
References
14
profiles in different leaf tissues and at different stages of
development. Nevertheless, enzymatic activity has
never been shown after heterologous expression (Clark
et al. 1997).
Geraniol also serves as a precursor for the biosynthesis of iridoid monoterpenoids and indole alkaloids
(El-Sayed and Verpoorte 2007; O’Connor and Maresh
2006; O’Connor and McCoy 2006). In connection
with these specialized plant metabolites, members of
the subfamily CYP76B, often wrongly named as
geraniol 10-hydroxylases, have been extensively
studied in Catharanthus roseus (CYP76B6) (Collu
et al. 2001; Sung et al. 2011) and Swertia mussotii
(CYP76B10) (Wang et al. 2010) and have been
demonstrated to catalyse the formation of 8-hydroxygeraniol (not 10-hydroxy-geraniol). Although Arabidopsis thaliana CYP76C10 has been granted a patent
as plant geraniol/nerol-8-hydroxylase (US patent No.
5753507) (Bak et al. 2011; Mizutani et al. 1997), true
function of the enzyme remains uncertain since neither
8-hydroxygeraniol nor iridoid derived secondary
products have been detected in vivo, yet. Lately, Sung
and co-workers demonstrated flavonoid 30 -hydroxylase function of CrCYP76B6; the enzyme is able to
specifically convert naringenin into eridictyol (Sung
et al. 2011). This finding was not only unexpected
because of the low sequence identity with other
flavonoid 30 -hydroxylases (35–37 % identity on
amino acid level), but also because of the different
locations of the biosynthesis of flavonoids and monoterpenoids in planta (Kaltenbach et al. 1999; Oudin
et al. 2007). Since the conversion of geraniol proceeds
approximately 10-times faster than naringenin’s and
Km values for geraniol are lower (Sung et al. 2011),
geraniol is likely to be the enzyme’s natural substrate.
The enzyme’s ability to transform naringenin might
solely be a consequence of the flexibility of the
substrate binding pocket, which is observed in vitro,
and thus reflect the high degree of function promiscuity that has been reported in both CYP76B and
CYP71A-families (Bozak et al. 1992; Hamberger and
Bak 2013; Robineau et al. 1998).
Monoterpenoid biosynthesis and cytochromes
P450 involved therein have been studied in-depth in
limonene biosynthesis in plants belonging to the mint
family (Lamiaceae). Both peppermint (Mentha x
piperita), spearmint (M. spicata), and perilla (Perilla
frutescens) express limonene hydroxylases, which
regiospecifically hydroxylate S-(-)-limonene at C3
Phytochem Rev (2015) 14:7–24
(CYP71D13 and CYP71D15), C6 (CYP71D18)
(Lupien et al. 1999) or C7-position (CYP71D174)
(Mau et al. 2010), respectively, thereby affording
(-)-E-isopiperitenol, (-)-E-carveol or perillyl alcohol.
High sequence identity between enzymes (70 %)
and the utilization of the same substrate allowed for
comprehensive studies of structure–function relationship (Schalk and Croteau 2000). The investigations
revealed that only a single amino acid controls
regiospecificity of CYP71D18, namely F363. Substitution with isoleucine converted the enzyme into a
regiospecifically conserved 6-hydroxylase. Reciprocal
mutation of the 3-hydroxylase (CYP71D15), however,
did not lead to a catalytically functional enzyme
(Schalk and Croteau 2000).
F363 is located 5 amino acids downstream of the
highly conserved ExxR-motif (see Figure 2 in Mau
et al. 2010) in substrate recognition site (SRS) 5 in the
loop between the K-helix and the b-sheet 1–4. Together
with SRS 1 and helix I, SRS 5 directly flanks the
substrate binding cavity. Recently, a different study
systematically investigated SRS 5 of more than 6,300
sequences (Seifert and Pleiss 2009). Independent of
Schalk and Croteau’s finding, also this study disclosed
the existence of a single amino acid residue that directs
substrate- and heme-interaction. It is situated at position
5 after the ExxR-motif and is often highly hydrophobic
(Seifert and Pleiss 2009; Sirim et al. 2010). Since this
motif faces towards the heme centre, it can be expected
to interact with all substrates during oxidation (Seifert
and Pleiss 2009). Thus, this position is of significant
interest for investigations with regards to structure–
function relationship of cytochromes P450 involved in
small molecule metabolism, and also for terpenoid
biosynthesis.
In other studies, structural analogues of the substrate S-(-)-limonene were used to analyse active site
interactions of CYP71D15 and CYP71D18. Besides
the wild types also all active mutants and chimera
exclusively provided either C3- or C6-specificity. This
implies that shape and rigidity of the active sites
restrict substrate orientation severely. Concerning the
structure of substrate analogues, investigations demonstrated that a methyl group at C1, bulkier substituents at C4, and additional carbon atoms at either C7
or C9 of limonene hamper catalytic activity. Thus,
deviation from the native limonene structure increases
loss of regioselectivity of CYP71D18 thereby suggesting steric interaction with the isopropenyl group in
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the natural substrate to be critical for the orientation in
the active site (Wust and Croteau 2002; Wust et al.
2001).
In contrast, CYP71D174 cloned from Perilla
frutescens shows less limitations in its product profile;
under optimal conditions, the enzymes hydroxylates
limonene affording a mixture of perillyl alcohol,
E-isopiperitenol and E-carveol. Consistent with
CYP71D18, CYP71D174 has a phenylalanine in the
position corresponding to the critical F363 discussed
before; a similar orientation of the substrate in the
active site was therefore assumed. Functional characterization of the heterologously expressed enzyme,
however, demonstrated that hydroxylation at C7 is
preferred upon C3 and C6, thus substrate orientation
must be controlled in a more complex way than by a
single amino acid residue. The outcome of these
experiments might be influenced by the fact that
heterologous expression of CYP71D174 was performed with a chimeric enzyme: to enhance enzyme
expression in E. coli, the first 10 N-terminal amino
acids were replaced by a modified bovine 17 alphahydroxylase. Thus, final conclusions can first be drawn
after heterologous expression of the native gene or
investigations in planta. Indeed, in vivo these findings
are not reflected, since the essential oil from the P.
frutescens variety originally used to clone the gene did
neither exhibit E-isopiperitenol nor E-carveol nor
traces or derived products thereof (Mau et al. 2010).
Recently, five new members of the CYP71Dsubfamily (CYP71D178–182) have been cloned from
oregano (Origanum vulgare) and thyme (Thymus
vulgaris). Comprehensive studies of CYP71D178,
CYP71D180 and CYP71D181—both in vitro and
in vivo—demonstrated their role in the biosynthesis of
the phenolic monoterpenes thymol and carvacrol.
Unlike limonene-hydroxylases of mint species, all
cytochromes P450 tested accepted a variety of different monoterpenes as substrates: c-terpinene, a-terpinene, (-)-R-a-phelladrene and (?)-R- as well as (-)-Slimonene. Results achieved rendered possible the
prediction of a reaction mechanism that proceeds via
an allylic alcohol intermediate instead of p-cymene.
With regards to structure–function relationship,
sequence comparisons do not indicate prominence of
the residue located five positions downstream of the
ExxR-motif. Just like CYP71D18, all three cytochromes P450 tested bear a phenylalanine residue at
this position. But their product profiles do not
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necessarily correlate to each other or with CYP71D18.
Instead, Crocoll and co-workers point at a residue two
amino acids downstream of this position with a marked
difference among the enzymes. Further investigations
including homology modelling and mutational studies
are necessary to clarify its significance (Crocoll 2011).
Another cytochrome P450 involved in monoterpene metabolism is CYP71A32 (menthofuran synthase) that catalyses conversion of (?)-pulegone to
(?)-menthofuran via allylic hydroxylation (Bertea
et al. 2001). This reaction can be regarded as unusual,
since the oxygen of the furan is not derived from the
original carbonyl oxygen but molecular oxygen
(Mizutani and Sato 2011). Especially under stressful
growth conditions (drought, high temperature, low
light intensity), menthofuran concentration in the
essential oil of peppermint can reach more than 5 %
which results in strong odour and off-colour on
storage. Thus, down-regulation of CYP71A32 has
been the focus of metabolic engineering in transgenic
peppermint plants (Mahmoud and Croteau 2001).
Domestication of strawberry (Fragaria ssp.) has led
to alterations in fruit flavour and aroma that are mainly
characterized by the presence or absence of certain
mono- and diterpenoids. Both wild and cultivated
strawberries express a pinene hydroxylase (PINH) in
ripe fruit and root tissue. Heterologously expressed,
this cytochrome P450, FaCYP71AR1, hydroxylates
a-pinene and limonene yielding myrtenol and perillyl
alcohol, respectively. It is mainly olefinic monoterpenes and myrtenyl acetate that contribute to the
volatile profile of wild strawberries (F. vesca). Perillyl
alcohol cannot be detected since its precursor is
lacking; the terpene synthase (FvPINS) of the wild
strawberry only produce linalool, a-pinene, b-myrcene and b-phellandrene, but no limonene (Mau and
Croteau 2006). The corresponding gene, however, is
defective in cultivated strawberry, and the terpene
synthase (FaNES1) catalyses the formation of the
sesquiterpenes nerolidol and linalool, thus establishing
the volatile profile of these fruits (Aharoni et al. 2004).
Finally a cytochrome P450 catalysing an atypical
reaction should be mentioned: CYP72A1, secologanin synthase from Catharanthus roseus, involved in
terpenoid indole alkaloid biosynthesis. The mechanism of reaction, C–C bond cleavage has been shown
to proceed through a radical mechanism (Irmler et al.
2000; Yamamoto et al. 2000), which is more closely
described in Mizutani and Sato (2011). First data
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suggested that the reaction takes place in the vacuole
(Contin et al. 1999), and it was assumed that it is the
lack of the proline-rich motif ([P/I]Px[P/G]xP), usually closely located to the N-terminal membrane
anchor, that prevents membrane-binding but targets
the enzyme to the vacuole instead. This speculation
was further supported by the fact that strictosidine
synthase, which in a subsequent step conjugates
secologanin and tryptamin, is localized in the vacuole
(Irmler et al. 2000). Yamamoto et al. (2000), in
contrast, demonstrated a tenfold higher enzyme
activity in the microsomal fraction of Lonicera
japonicus-suspension cultures cells than in the crude
extract, thereby indicating that the enzyme is rather
membrane-associated.
CYP72A1 is atypical in another way: it is the only
cytochrome P450 not belonging to CYP71 clan so far
to be described being involved in biosynthesis of
specialized terpenoids, and thus is derived from a
different ancestoral gene than all other cytochrome
P450 described in this publication (see Fig. 1).
Cytochromes P450 involved in sesquiterpene
biosynthesis
So far, less than ten cytochromes P450 mediating steps
in sesquiterpene biosynthesis have been thoroughly
investigated after cloning and heterologous expression. Within these, an unexpected substrate promiscuity as well as flexibility regarding regiospecificity
has been observed.
One of the first cytochrome P450 involved in
sesquiterpene biosynthesis studied was 5-epi-aristolochene 1,3-dihydroxylase (CYP71D20) that plays a
decisive role in capsidiol biosynthesis in Nicotiana
tabacum (Solanaceae). Since the transformation of
5-epi-aristolochene to capsidiol requires two hydroxylation reactions that are both specific with regards to their
regio- and stereospecificity, at first, catalysis through a
single enzyme was not expected. Also, the mechanism
of reaction remained unknown for a long time since the
existence of monohydroxylated intermediates could not
be proven (Ralston et al. 2001). Instead, it was suggested
that the monohydroxylated intermediate rotated within
the active site. Study of CYP71D20’s structure–function relationship, accomplished by molecular modelling
and targeted mutation, have eventually demonstrated
that the preferred reaction order of hydroxylation is at
Phytochem Rev (2015) 14:7–24
C-1 followed by C-3. It remains to be shown whether the
intermediate is released in between the reactions
(Takahashi et al. 2005).
Coupled assays with CYP71D20 and the sesquiterpene synthases germacrene A synthase and premnaspirodiene synthase, respectively, revealed that
hydroxylation of germacrene A proceeds at two
different positions, whereas premnaspirodiene is
hydroxylated twice at position 2, the latter resulting
in the corresponding ketone, solavetivone (Greenhagen et al. 2003). In planta data are missing, but needed
in order to demonstrate, whether the outcome of these
experiments indeed reflect endogenous enzyme activities or whether the observations were affected by the
artificial environment of the in vitro-tests. Investigations into the relationship between structure and
function of CYP71D20 focused on two amino acids:
serine368 and isoleucine486. Ser368 is located 5 amino
acids after the highly conserved ExxR-motif, a
position previously demonstrated to be critical for
substrate-enzyme interaction (cp. CYP71D18). Ile486
occupies a position in SRS 6 that has been found to be
highly variable. Targeted-mutation of these residues
indicated that both positions are crucial for regio- and
stereospecificity as well as regulation of the catalysed
reactions (Takahashi et al. 2005).
Conclusions achieved in these studies prove incoherent when CYP71D55, premnaspirodiene oxygenase from Hyoscyamus muticus (Solanaceae), was
examined. Besides its native substrate premnaspirodiene, CYP71D55 is capable of hydroxylating valencene, 5-epi-eremophilene, and 5-epi-aristolochene.
The C-2 atom of premnaspirodiene is hydroxylated
twice yielding solavetivone. In contrast, 5-epi-eremophilene and 5-epi-aristolochene are only hydroxylated
once. Comparison of CYP71D20 and CYP71D55
revealed high identity on amino acid level. Within
domains relating to SRS 5 and 6, differences in only 4
amino acids were observed. Comprehensive mutational studies on these 4 residues were conducted both
in CYP71D20 and CYP71D55. The experiments
demonstrated that these 4 residues indeed influence
catalytic efficiency but do not lead to alternation of
regio- or stereospecificity, the latter was evident in
case of 5-epi-aristolochene that is hydroxylated at
different positions by CYP71D20 and CYP71D55,
respectively (see Table 2). The regiospecificity of
CYP71D55 appears independent of the overall hydrocarbon scaffold (spirovetivane vs. eremophilane).
17
Stereochemistry, however, seems to be best predicted
by the orientation of the vicinal methyl at C-14 and
C-15 instead of the position of the double bond in the A
or B ring, since the vicinal methyl groups guides the
cytochrome P450 in the opposite stereochemical
direction. For the reaction mechanism, it is likely that
the two successive hydroxylations are undertaken
independently, but do not necessarily depend on a
dissociation of the first substrate (Takahashi et al.
2007). Since only solavetivol but not nootkatol is
converted to the corresponding ketone, it can be
presumed that the active site is restricted, although
further studies are needed in order to elucidate these
promiscuous enzymes.
Within cotton plants (Gossypium arboreum, Malvaceae), most sesquiterpenoids derive from d-cadinene.
One of these is gossypol, a polyphenol with high
antioxidative activity. In the plant, gossypol serves as a
defence compound by causing infertility in most male
animals including humans. Therefore, gossypol was
tested as a contraceptive for men (Coutinho 2002).
Apart from the sesquiterpene synthase that catalyses
formation of cadinene, only an O-methyl transferase
and a cytochrome P450 involved in gossypol’s biosynthesis have been described. CYP706B1 (part of the
CYP71 clan) catalyses hydroxylation of d-cadinene at
C8 (Luo et al. 2001). To date, CYP706B1 is the only
member of the CYP706-family with assigned function
(Nelson and Werck-Reichhart 2011). CYP706B1’s
catalytical activity was studied towards compounds
present in cotton glands (a-humulene and a-copaene)
and structurally related compounds (a-cubebene and amuurolene). All substrates were hydroxylated once with
the exception of a-copaene, which was not transformed.
In several cases, generation of up to four products was
reported; exact structures of the products have not been
published (Wang and Essenberg 2010).
The cytochrome P450 CYP71BA1 from Zingiber
zerumbet (Zingiberaceae) catalyses the conversion of
a-humulene to 8-hydroxy-a-humulene in the zerumbone biosynthesis. After heterologous expression of
the gene in yeast, formation of this single product was
described (Yu et al. 2011). Therefore, it was proposed
that the subsequent and final step of zerumbone
biosynthesis was mediated by an alcohol dehydrogenase, whose elucidation was reported in a recent
publication (Okamoto et al. 2011).
Sesquiterpene lactones are characteristic products of
species in the large plant families Asteraceae and
123
123
71AV8
GAO
Artemisia
annua
71AV1
(Asteraceae)
Cichorium
intybus
(all:
Asteraceae)
Barnadesia
spinosa
Helianthus
annuus
OH
H
O
HO
O
It also catalyses hydroxylation of germacrene A and amorphadiene-4,11-diene affording artemisinic acid and germacrene A acid, respectively (see CYP71AV1
and GAO)
germacrene A acid
HQ166835
GU256647
GU256646
GU256645
Saussurea
costus
artemisinic acid OH
O
GU256644
H
O
Cankar
et al.
(2011)
Nguyen
et al.
(2010)
Ro et al.
(2008),
Teoh
et al.
(2006)
DQ315671
Cichorium
intybus
germacrene A
OH
References
Database
number
GU198171
amorpha-4,11-diene
Catalysed
eaction
Lactuca
sativa
(Asteraceae)
Plant
origin
Cytochrome
P450
Table 2 Cytochrome P450 genes involved in sesquiterpene biosynthesis
18
Phytochem Rev (2015) 14:7–24
71D20
71BL3
71BL2
71BL1
Zingiber zerumbet
71BA1
(Solanaceae)
Nicotiana tabacum
(Asteraceae)
Cichorium intybus
(Asteraceae)
Lactuca sativa
(Asteraceae)
Helianthus annuus
(Zingiberaceae)
Plant
origin
Cytochrome
P450
Table 2 continued
Germacrene A and spirodiene are also hydroxylated, but only with (very) low efficiency
Catalysed
eaction
AF368376
JF816041
HQ439599
Greenhagen et al. (2003), Ralston et al. (2001), Takahashi et al. (2005)
Liu et al. (2011)
Ikezawa et al. (2011)
Ikezawa et al. (2011)
Yu et al. (2011)
AB331234
HQ439587
References
Database
number
Phytochem Rev (2015) 14:7–24
19
123
123
706B1
Hyoscyamus
muticus
71D55
(Malvaceae)
Gossypium
arboreum
(Solanaceae)
Plant
origin
Cytochrome
P450
Table 2 continued
Additional substrates are (-)-d-cadinene, (-)-a-cubebene, (-)-a-muurolene, a-humulene, the structure of the respective products
has not been described
Catalysed
eaction
Luo et al. (2001), Wang and Essenberg
(2010)
Takahashi et al. (2007)
EF569601
AF332974
References
Database
number
20
Phytochem Rev (2015) 14:7–24
Phytochem Rev (2015) 14:7–24
Apiaceae. Many of them are not only of importance for
the plants, but also exhibit social and economic value to
mankind. Artemisinin, helenanin, matricin and thapsigargin exhibit curative effects that are applied for the
treatment of diseases both in conventional and traditional medicine (Drew et al. 2009; Ramadan et al. 2006;
Ro et al. 2006), nootkatone can be used as flavour and
fragrance ingredient by the food industry (Berry et al.
1967), to mention only a few of them. Thus, interest in
the biosynthesis of these compounds is high.
The discovery of CYP71AV1, amorpha-4,11-diene
oxidase of Artemisia annua, rendered possible the
biotechnological synthesis of an artemisinin precursor
for the first time. Catalysed by the enzyme’s activity,
amorpha-4,11-diene is transformed into artemisinic
acid in three consecutive hydroxylation steps (Ro et al.
2006). Even though artemisinic acid does not serve as
artemisinin precursor in planta (Bertea et al. 2005; Teoh
et al. 2009), it is a valuable compound for semisynthetic
artemisinin production. CYP71AV1 has additionally
been studied in planta. Examinations of its expression in
Nicotiana species have been published recently by two
groups. In N. benthamiana, co-expression of amorpha4,11-diene synthase (ADS) and CYP71AV1 led to the
formation of artemisinic acid, which was partly recovered as diglucoside (van Herpen et al. 2010). The group
of Brodelius, on the other hand, only obtained amorpha4,11-diene and artemisinic alcohol when expressing the
same enzymes in N. tabacum. This finding was
confirmed studying the enzymes in Artemisia annua
(Olofsson et al. 2011). Studies on expression and
product formation of the two dehydrogenases (alcohol
and aldehyde) along with CYP71AV1 demonstrated
that CYP71AV1 can catalyse the production of both the
alcohol and the aldehyde, but also that several endogenous A. annua dehydrogenases can catalyse the
production of the acid and the aldehyde (Li et al.
2012; Olofsson et al. 2011).
Subsequently, CYP71AV-genes were isolated from
seven more Asteraceae species (CYP71AV2–8).
Indeed, the CYP71AV-subfamily is conserved in all
major aster subfamilies and might even be highly
specific for sesquiterpene lactone biosynthesis (Teoh
et al. 2006). With the exception of CYP71AV1, all
other subfamily members are germacrene A oxidases
(GAO) with exceptional broad substrate specificity.
In vitro, GAOs have been shown to mediate hydroxylation of amongst others germacrene A, valencene
and amorpha-4,11-diene to the corresponding acids
21
(Cankar et al. 2011; Nguyen et al. 2010). Since
hydroxylation does not proceed regiospecifically, the
hypothesis of two distinct categories of sesquiterpene
modifying cytochromes P450 was disproved: a division between those acting on the A-ring like
CYP71D55 and CYP71D20 and those that act on
allylic C12 as CYP71AV1 cannot further be sustained.
Based on these observations, CYP71AV-subfamily
has been chosen for the study of enzyme evolution. It
has been assumed that substrate promiscuity provides
selective advantage for an enzyme, allowing optimization of one specific function under specific selection
pressure as observed in Artemisia annua: mutations
both in its terpene synthase as well as GAO gave rise to
unique artemisinin biosynthesis (Nguyen et al. 2010).
Members of a novel CYP71-subfamily mediate
hydroxylation of germacrene A acid at positions
adjacent to the carboxy group. CYP71BL2 from
Lactuca sativa (Ikezawa et al. 2011) and CYP71BL3
from Cichorium intybus (Liu et al. 2011) specifically
catalyse 6a-hydroxylation. Subsequently, the intermediate undergoes spontaneous lactone formation that
results in costunolide. Homologues of CYP71BL2 have
been found in the EST databases of all major Asteraceae
subfamilies except of the Helianthae tribe implying that
costunolide biosynthesis is conserved in most Asteraceae genera. Instead, Helianthae possess ESTs highly
homologue to CYP71BL1, an 8b-hydroxylase that has
been isolated from Helianthus annuus. In case of 8bhydroxylation, no spontaneous lactone formation has
been observed due to the physiochemical properties of
the molecule, which show that the lactone 8,12guaianolides need a yet non-disclosed enzyme in order
to be formed. So far, comparative genomics analysis
suggests that Asteraceae either possess a homologue to
the 6a- or the 8b-hydroxylase, but not both of them.
This does, however, not correlate with existence or
absence of sesquiterpene lactones of the costunolidetype (Ikezawa et al. 2011).
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