Evident and latent plasticity across the rice diterpene synthase family with potential implications for the evolution of diterpenoid metabolism in the cereals

Article abstract of DOI:10.1042/BJ20101429

The evolution of natural product biosynthetic pathways can be envisioned to occur via a number of mechanisms. In the present study we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-CPP (copalyl diphosphate), which are substrates for downstream diterpene synthases. In the present paper we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent-, syn- or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported ent-sandaracopimaradiene synthase also converts syn-CPP into syn-labda-8(17),12E,14-triene, which can be found in planta. Notably, normal CPP is not naturally found in rice. Thus the presence of diterpene synthases that react with this non-native metabolite reveals latent enzymatic/metabolic plasticity, providing biochemical capacity for utilization of such a novel substrate (i.e. normal CPP) which may arise during evolution, the implications of which are discussed. The Authors Journal compilation

Full text of DOI:10.1042/BJ20101429

Biochem. J. (2011) 435, 589–595 (Printed in Great Britain) doi:10.1042/BJ20101429
589
Evident and latent plasticity across the rice diterpene synthase family with
potential implications for the evolution of diterpenoid metabolism in the
cereals
Dana MORRONE, Matthew L. HILLWIG, Matthew E. MEAD, Luke LOWRY, D. Bruce FULTON and Reuben J. PETERS1
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, U.S.A.
The evolution of natural product biosynthetic pathways can be
envisioned to occur via a number of mechanisms. In the present
study we provide evidence that latent plasticity plays a role in such
metabolic evolution. In particular, rice (Oryza sativa) produces
both ent- and syn-CPP (copalyl diphosphate), which are substrates
for downstream diterpene synthases. In the present paper we
report that several members of this enzymatic family exhibit
dual reactivity with some pairing of ent-, syn- or normal CPP
stereochemistry. Evident plasticity was observed, as a previously
reported ent-sandaracopimaradiene synthase also converts syn-
CPP into syn-labda-8(17),12E,14-triene, which can be found
in planta. Notably, normal CPP is not naturally found in rice.
Thus the presence of diterpene synthases that react with this non-
native metabolite reveals latent enzymatic/metabolic plasticity,
providing biochemical capacity for utilization of such a novel
substrate (i.e. normal CPP) which may arise during evolution, the
implications of which are discussed.
Key words: metabolic evolution, phytoalexin, substrate
specificity, terpene synthase, terpenoid natural product.
has long been appreciated that it produces at least 20 such
natural products as antimicrobial phytoalexins, and relatively
early sequencing of the rice genome has led to functional
characterization of the corresponding diterpene synthase gene
families [4,6]. However, since rice produces only ent- and
syn-CPP, as first suggested by substrate studies with cell-free
enzyme assays [7,8], and then more conclusively demonstrated
by functional genomics-based investigations of the rice CPS
family [9–11], the rice KSL (OsKSL) was only assayed
with these two stereoisomers [12–18]. This OsKSL family, with
eight functional members (Scheme 2), provided an opportunity
to comprehensively explore diterpene synthase substrate
stereospecificity not only for individual enzymes, but also
across an entire organism/genome, providing insight into the
encoded metabolic plasticity. Accordingly, we have utilized our
modular metabolic engineering system [19] to investigate the CPP
substrate stereospecificity of the OsKSL family. The results of
this substrate specificity screen revealed not only evident, but
also latent, metabolic plasticity, as well as offering some insight
into diterpene synthase substrate recognition, all of which are
discussed below.
INTRODUCTION
Terpenoids, with over 40000 known individual compounds,
form the largest class of natural products [1]. Prominent among
these are the ∼7000 labdane-related diterpenoids, which are
particularly prevalent in plants, where the affiliated gibberellin
phytohormone production required for normal growth and
development provides a genetic reservoir that has been repeatedly
drawn upon for the evolution of such novel natural products [2,3].
The unifying biogenetic feature of labdane-related diterpenoid
biosynthesis is its initiation by a sequential pair of cyclization
and/or rearrangement reactions, and the corresponding enzymes
are often found as functionally diverse gene families in plants
[4,5]. The characteristic decalin ring structure is formed from the
universal diterpenoid precursor GGPP [(E,E,E)-geranylgeranyl
diphosphate] by initially acting class II diterpene cyclases,
which most commonly produce a labdadienyl/CPP (copalyl
diphosphate) of normal (9S,10S), syn (9S,10R) or ent (9R,10R)
configuration (Scheme 1), with the corresponding enzymes
termed CPSs (CPP synthases). The resulting CPP is subsequently
utilized by class I diterpene synthases, which lyse the allylic
diphosphate ester linkage to initiate carbocationic cyclization
and/or rearrangement reactions. Because the corresponding
enzyme in gibberellin biosynthesis is KS (ent-kaurene synthase),
the derived class I diterpene synthases are often termed KSL
(KS-like). However, although these enzymes do not react with
GGPP and are typically assumed to be specific for particular
stereoisomers of CPP, comprehensive investigation of such
substrate stereospecificity (e.g. with all three commonly found
CPP stereoisomers) has not been reported.
EXPERIMENTAL
General
Unless otherwise noted, chemicals were purchased from Fisher
Scientific, and molecular biology reagents were from Invitrogen.
All recombinant expression was carried out using the OverExpress
C41 strain of Escherichia coli (Lucigen), which is well-suited to
T7 promoter-based expression of plant-derived labdane-related
diterpene synthases [20]. GC-MS was performed with a Varian
Rice (Oryza sativa) has become a model system for
investigation of labdane-related diterpenoid biosynthesis, as it
Abbreviations used: CPP, copalyl diphosphate; CPS, CPP synthase; CYP, cytochrome P450 mono-oxygenase; DQF-COSY, double-quantum-filtered
correlation spectroscopy; FID, flame ionization detection; GGPP, (E,E,E)-geranylgeranyl diphosphate; GST, glutathione transferase; HMBC, heteronuclear
multiple bond correlation; HSQC, heteronuclear single-quantum coherence; KS, ent-kaurene synthase; KSL, KS-like; MEP, methyl erythritol phosphate;
OsKSL, rice (Oryza sativa) KSL.
1
To whom correspondence should be addressed (email rjpeters@iastate.edu).
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then shifted to 16◦C for 1 h prior to induction with 0.5 mM
IPTG (isopropyl β-D-thiogalactopyranoside), with the cultures
then fermented for an additional 72 h at 16◦C with continued
shaking. The cultures were then extracted with an equal volume
of hexane, with 1:50 (v/v) ethanol added to disrupt emulsions.
Diterpene production was quantified by GC-FID (flame ionization
detection) analysis, with GC-MS comparison with authentic
standards enabling identification of all resulting compounds but
that from co-expression of OsKSL10 with pGGsC (i.e. syn-CPP).
To directly determine the structure of the OsKSL10 product
from syn-CPP, we utilized previously described methodology to
improve production levels in our metabolic engineering system
[23], which enabled facile generation of the milligram quantities
needed for structural characterization by NMR spectroscopy.
Specifically, we co-transformed pDEST15/OsKSL10 and pGGsC
with the further co-compatible pIRS plasmid that increases flux
into the endogenous MEP (methyl erythritol phosphate) pathway
by overexpression of the idi, dxs, and dxr genes from E. coli.
The resulting recombinant bacteria were grown and extracted
as described above, but as a 1 litre culture supplemented with
the MEP pathway precursors pyruvate (50 mM) and 2% (v/v)
glycerol. The resulting 1 litre of hexane extract was reduced to
∼10 ml by rotary evaporation, passed over a ∼5 ml silica-gel
column to remove any polar contaminants, and then dried under
nitrogen gas, yielding ∼15 mg of the unknown diterpene, which
was then re-dissolved in 0.4 ml of C2HCl3 for NMR analysis.
Scheme 1 Cyclization of GGPP to stereoisomeric CPP by CPS
Note that the position of the C5 hydrogen is always anti to the C10 methyl group due to the
chair–chair conformation of the GGPP in this initial CPS-catalysed (i.e. class II) cyclization
mechanism.
3900 GC with Saturn 2100 ion-trap mass spectrometer in electron
ionization (70 eV) mode using a HP-5ms column. Samples
(1 μl) were injected in splitless mode at 50◦C and, after holding
for 3 min at 50◦C, the oven temperature (unless otherwise noted)
was raised at a rate of 14◦C/min to 300◦C, where it was held for
an additional 3 min. MS data from 90 to 600 m/z were collected
starting 12 min after injection until the end of the run.
Functional characterization via metabolic engineering
Functional characterization of the substrate stereospecificity
of the OsKLS was accomplished by use of our previously
described modular metabolic engineering system [19]. Briefly,
using this system it is possible to co-express class I labdane-
related diterpene synthases such as the OsKSL from pET-based
pDEST expression vectors with GGPP and CPS carried on
a compatible pACYC-Duet (Novagen/EMD)-derived plasmid,
where pGGnC carries the D621A mutant of abietadiene synthase
from Abies grandis that produces CPP of normal stereochemistry
[21], while pGGeC carries the An2/ZmCPS2 ent-CPS from
Zea mays [22], and pGGsC carries the OsCPS4 syn-CPS from
rice [10]. Accordingly, C41 cells were transformed with the
appropriate plasmid enabling functional recombinant expression
of each OsKSL, as described previously [18], along with
one of the three pGGxC vectors described above, in all possible
pairings. These recombinant bacteria were then grown in 20 ml
of liquid media cultures with shaking at 37◦C to A600∼1.0,
Chemical structure identification
NMR spectra for the unknown syn-CPP-fed OsKSL10 pro-
duct were recorded at 25◦C on a Bruker Avance 500
spectrometer equipped with a cryogenic probe for 1H and
13C. Structural analysis was performed using 1H, one-
dimensional, DQF-COSY (double-quantum-filtered correlation
spectroscopy), HSQC (heteronuclear single-quantum coherence),
HMQC (heteronuclear single quantum correlation), HMBC
(heteronuclear multiple bond correlation) and NOESY (nuclear
Overhauser enhancement spectroscopy) experiment spectra
acquired at 500 MHz, and 13C and DEPT135 spectra (125.5
MHz) using standard experiments from the Bruker TopSpin
v1.3 software. Chemical shifts were referenced to the known
Scheme 2 Rice diterpene metabolic map [17,18]
Shown are the reactions catalysed by the various rice diterpene cyclases [both class II CPS, and class I KS(L)].
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Table 1 1H and 13C NMR assignments for syn-labda-8(17),12E,14-triene
the known starting concentration of substrate to calculate the
corresponding catalytic rate.
C
δC (p.p.m.)
δH (p.p.m.), multiplicity
J (Hz)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
37.16
19.39
42.92
33.48
46.04
23.85
31.88
149.23
58.42
38.25
25.89
133.36
133.68
142.01
110.14
12.12
109.72
33.78
22.45
22.40
1.086 (m), 1.550 (m)
1.434 (m), 1.607 (m)
1.167 (dt), 1.403 (m)
Extraction of rice leaves
Rice leaves that had been treated with methyl jasmonate for
36 h, as described previously [10], were frozen at − 80◦C.
Approximately 100 g of these frozen leaves were cut up and
then homogenized by grinding in a mortar and pestle under
liquid nitrogen in the presence of glass beads. After grinding,
the resulting powder was extracted with 75 ml of hexanes, then
75 ml of ethyl acetate, and finally 75 ml of tetrahydrofuran. The
combined organic extracts were reduced by rotary evaporation,
passed over silica gel, and dried under nitrogen as described
above. The resulting material was then resuspended in 1 ml of
ethyl acetate, with 20 μl aliquots being removed and then dried
under nitrogen, and resuspended in 20 μl of hexane for analysis
by GC-MS, using an extended temperature ramp where, after the
initial 3 min hold at the 50◦C injection temperature, the oven
temperature was raised at 18◦C/min to 190◦C, then slowed to
2.5◦C/min until reaching 275◦C, following which it was raised
at 20◦C/min to 300◦C, where it was held for 2 min.
3.6, 13.1
1.298 (m)
1.296 (m), 1.611 (m)
2.065 (m), 2.146 (m)
1.635 (m)
2.164 (m), 2.409 (dt)
5.365 (t)
15.0, 5.6
7.1
6.330 (dd)
4.864 (d), 5.020 (d)
1.707 (s)
4.474 (t), 4.640 (t)
0.875 (s)
0.800 (s)
10.7, 17.4
10.7, 17.4
1.6, 2.2
0.909 (s)
chloroform-d (13C 77.23, H 7.24 p.p.m.) signals offset from
TMS (trimethylsilyl). Correlations from the HMBC spectra were
used to propose a partial structure, whereas connections between
protonated carbons were obtained from DQF-COSY to complete
the partial structure and assign proton chemical shifts. The
configuration of the A and B rings is predetermined by the con-
figuration of the CPP enzyme substrate, since chemical bonds in
that portion of the molecule are not altered. The configuration of
the acyclic portion of the molecule (C11–C16) was determined
by reference to spectra for normal labdatrienes, which have been
previously characterized [24]. Specifically, the C12–C13 double-
1
RESULTS
Screening OsKSL substrate specificity
To investigate the substrate stereospecificity of the entire OsKSL
family, we turned to our previously developed modular metabolic
engineering system [19]. In particular, using this system it
was possible to co-express each of the OsKSLs with a GGPP
synthase and CPS producing one of the three commonly found
stereoisomers of CPP (note that all of the OsKSLs have been
demonstrated to not react with the acyclic GGPP precursor).
Accordingly, the ability of each of the eight active OsKSLs
to react with CPP of ent-, syn- or normal stereochemistry was
determined by simply extracting the corresponding recombinant
bacterial cultures and analysing the resulting organic extract for
the presence of diterpenes by GC-MS. The previously reported
activity for each of the OsKSLs, as summarized in Scheme 2,
was evident. In addition, while the other OsKSLs were specific
for a single stereoisomer of CPP, OsKSL4 and OsKSL11 reacted
with CPP of normal stereochemistry along with their previously
reported activity with syn-CPP, and OsKSL10 was active with
syn-CPP as well as its previously reported activity with ent-CPP.
Surprised by this latter result, we purified OsKSL10 and verified
its reactivity with syn-CPP, with the same product resulting from
in vitro reactions as found in the context of the bacterial metabolic
engineering system. In all three cases, diterpene yield with the
‘alternative’ CPP stereoisomer approached that found with
the ‘native’ stereoisomer, being limited to less than 3-fold
decreases. As yield in our metabolic engineering system has been
correlated with catalytic efficiency [25,26], this suggests that these
enzymes exhibit reasonable affinity for and activity with their
‘alternative’ substrates.
bond configuration was determined to be E based on the C14 13
C
chemical shift for syn-labda-8(17),12E,14-triene (142.01 p.p.m.).
The comparison of E and Z configuration of normal labda-
8(17),12,14-trienes shows a dramatic ∼10 p.p.m. difference in
the C14 chemical shift in E compared with Z configurations
[∼131 p.p.m. (Z) compared with ∼141 (E) p.p.m.]. This reference
data and our NMR analysis support the presented syn-labda-
8(17),12E,14-triene double bond configuration for the OsKSL10
1
product. The assignments for the annotated 13C and H one-
dimensional spectra are presented in Table 1.
Enzymatic assays
Assays were performed with the purified GST (glutathione
transferase)–OsKSL10 fusion protein using enzymatic coupling,
as described previously [25,26]. Briefly, GGPP was completely
converted into either ent- or syn-CPP by incubation with the
appropriate CPS for 5 h at room temperature (22◦C). Assays
were performed in duplicate with various concentrations of GGPP
ranging from 0.1 μM to 100 μM, with time and GST–OsKSL10
concentration varied to ensure that measurements were taken
in the linear portion of the reaction progress curve. The assays
were inactivated by heating the solution to 65◦C for 5 min, with
the remaining substrate then dephosphorylated by treatment with
CIP (calf intestinal phosphatase; New England Biolabs), enabling
extraction of both the diterpene product and copalol (derived from
dephosphorylation of CPP). This was done to enable correction
for handling errors by measurement of the fractional turnover
(i.e. comparison of the relative amounts of diterpene product
and copalol by GC-FID), which was used in combination with
Alternative substrate product identification
By comparison with available authentic standards, OsKSL4
activity with CPP of normal stereochemistry was found to
produce a mixture of ∼90% pimara-8(14),15-diene and ∼10%
isopimara-7,15-diene, whereas OsKSL11 reacted with the same
substrate to yield essentially only isopimara-8(14),15-diene (also
known as sandaracopimaradiene; Figure 1). However, it was not
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Figure 1 Identification of alternative products by comparison with authentic standards
(A) GC-MS-selected ion chromatogram of OsKSL4 products from normal CPP. (B) Mass spectra of OsKSL4 major product. (C) Mass spectra of authentic pimara-8(14),15-diene. (D) GC-MS-selected
ion chromatogram of OsKSL11 products from normal CPP. (E) Mass spectra of OsKSL11 major product. (F) Mass spectra of authentic sandaracopimaradiene.
possible to identify the product of OsKSL10 activity with syn-CPP
in this fashion, as it did not match any authentic standard on-hand.
This diterpene was then identified by increasing flux towards
terpenoid production, via up-regulation of the endogenous
isoprenoid precursor supply pathway as described previously [23],
to enable the corresponding recombinant bacteria to produce
enough of the novel product for NMR analysis (∼15 mg,
following purification from 1 litre of bacterial culture). The
structure was assigned by analysis of collected HMBC, HSQC
and COSY spectra, and found to be syn-labda-8(17),12E,14-triene
(Table 1), completing the assignment of the observed alternative
reactions (Scheme 3).
the possibility that OsKSL10 produces syn-labdatriene in planta
was examined by phytochemical analysis of methyl-jasmonate-
induced and uninduced rice leaves. As previously reported [7],
extracts from control (uninduced) rice leaves contained only
small amounts of ent-kaurene, whereas numerous diterpenes
were detected in extracts from induced rice leaves (Figure 2).
These specifically included syn-labdatriene, as well as syn-
stemodene, whose presence in planta had not been previously
demonstrated, although the OsKSL11 responsible seems widely
conserved [17]. Steady-state kinetic analysis was also consistent
with physiological relevance of the ability of OsKSL10 to react
with both ent- and syn-CPP, as it exhibited similar catalytic
efficiency with each (Table 2). Notably, consistent with the lack
of a normal CPP producing CPS in the rice genome, whereas the
syn-CPP-derived products of OsKSL11 (as noted above), as well
as OsKSL4, were readily observed, the pimaradienes produced
by these diterpene synthases from normal CPP were not evident.
Physiological relevance of syn-labdatriene
Because rice produces both ent- and syn-CPP, it seemed
possible that the dual substrate reactivity of OsKSL10 was
physiologically relevant (i.e. both products can be found in
planta). The labdane-related diterpenoid natural products of rice
are largely thought to be anti-microbial phytoalexins [4,6], whose
biosynthesis, by definition [27], is induced as part of the plant
microbial defence response. This includes transcriptional up-
regulation of OsKSL10, as well as OsKSL4 [14]. Accordingly,
DISCUSSION
On the basis of the genome sequence available [28], the entire
family of rice diterpene synthases is known, and subsequent
biochemical characterization has assigned unique functional roles
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Scheme 3 Alternative reactions catalysed by (A) OsKSL4, (B) OsKSL11 and
(C) OsKSL10
Table 2 OsKSL10 steady-state kinetic constants
Substrate
Km ( μM)
kcat (min− 1
)
kcat/Km (M− 1 · s− 1
)
5.6 × 103
1.7 × 103
+
+
−
ent-CPP
syn-CPP
0.6 0.1
0.02 0.01
0.05 0.02
−
−
+
−
+
5
1
Figure 2 Detection of syn-labda-8(17),12E,14-triene in induced rice plant
tissue
to each of these [15,18]. Hence it is possible to define this early
part of the labdane-related diterpene metabolic network for this
important crop plant (Scheme 2), with the resulting/downstream
natural products largely thought to be involved in the microbial
defence response of the plant [4,6]. One outcome of the studies
reported in the present paper is the addition of the syn-labdatriene
product of OsKSL10 to this metabolic network. Given the
inducible nature of the transcription of OsKSL10 [14], and the
role of its already known ent-sandaracopimaradiene product as
a precursor for the antifungal phytoalexins oryzalexins A–F, it
seems likely that any syn-labdatriene-derived natural products
will similarly function as antifungal agents, although this remains
to be demonstrated. This would be consistent with similar
transcriptional induction of all of the other diterpene synthases
involved in biosynthesis of the >20 known rice diterpenoid
phytoalexins [4,6,9–16]. Nevertheless, one intriguing alternative
possibility is a role for syn-labdatriene in the production of an
inducible signalling molecule, as has been previously indicated
for a similar labdatriene-derived diterpenoid in tobacco [29]. On
the other hand, the ability of OsKSL4 and OsKSL11 to react with
normal CPP is not directly relevant in rice, whose genome does
not contain a CPS that produces this stereoisomer [9–11].
(A) GC-MS-selected ion chromatogram of induced rice leaf extract (* indicates the peak
corresponding to syn-stemodene product of OsKSL11). (B) Mass spectrum of 15.53 min
peak in induced rice leaf extract. (C) Mass spectrum of authenticated syn-labda-
8(17),12,14-triene (retention time = 15.51 min).
obvious structural similarity between these two stereoisomers is
the occurrence of co-axial β-methyl substituents on C4 and C10,
which provides steric bulk on this ‘upper face’ of the trans-decalin
nucleus (Figure 3). Presumably, it is this common feature that
enables the dual reactivity exhibited by OsKSL4 and OsKSL11.
In addition, the stereochemistry of the observed products is
consistent with similar conformations of both substrates in each
active site (Figure 4). By contrast, the ability of OsKSL10 to react
with both ent- and syn-CPP presumably arises from the common
C9α connection of the isoprenyl-diphosphate-containing ‘tail’ to
the substrate nucleus (Figure 3), although the lack of cyclization
with syn-CPP prevents any insights into the relative conformation
of these two substrates within the active site.
Notably, some plasticity in substrate specificity has been
previously observed in other terpene synthases, leading to
suggestions of evolutionary relevance. Perhaps most strikingly,
work in strawberries has demonstrated a clear evolutionary
derivation of a linalool/nerolidol synthase in cultivated strawberry
from monoterpene synthases found in wild strawberry plants,
wherein the loss of the plastid targeting pre-sequence from a
In any case, the ability of certain rice diterpene synthases to
react with two different stereoisomers of CPP raises the question
of how such dual substrate recognition is achieved. OsKSL4
and OsKSL11 react with both syn- and normal CPP, and the
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angiosperms, with no obvious homology between the relevant
known enzymes from these two broad groups [5,32–34], it
seems likely that neofunctionalization of the corresponding class
II diterpene cyclases to production of this CPP stereoisomer
occurs with some appreciable frequency. Indeed, recent results
demonstrate that the closely related monocot cereal crop plant
wheat (Triticum aestivum) produces normal rather than syn-
CPP (Y. Wu, T. Toyomasu and R.J. Peters, unpublished work).
Accordingly, the latent metabolic plasticity evident in the
present study from the substrate promiscuity of these two
rice diterpene synthases provides a means by which such
emergence of stereochemically differentiated substrates could be
immediately incorporated into ‘secondary’ metabolism, which
seems pertinent to such biochemical evolution in the cereal
plant family. In particular, coupled with the often-observed
substrate plasticity of downstream tailoring enzymes, especially
CYPs (cytochrome P450 mono-oxygenases), this would then
enable emergence of essentially complete biosynthetic pathways
(e.g. the bioactive oryzalexins D–F are simply diterpenoid diols
only two hydroxylation reactions removed from their diterpene
precursor). For example, rice contains ∼350 CYPs [35], with
the large CYP71 and CYP76 families (74 and 28 members
respectively) each containing members involved in labdane-
related diterpenoid biosynthesis [36,37]. Notably, at least one of
these can hydroxylate pimaradienes of normal stereochemistry
(Q. Wang, M.L. Hillwig, S. Swaminathan and R.J. Peters,
unpublished work). Thus, provided access to a novel metabolite
substrate, the latent plasticity of terpene synthases, such as
that shown in the present study, can immediately give rise to
potentially bioactive natural products, providing a mechanism
for rapid evolution of novel biosynthetic pathways. Although
not directly selectable in and of itself, such latent plasticity
would be periodically uncovered upon the introduction of novel
metabolites, presumably providing a corresponding selectable
advantage, albeit in punctuate fashion.
Figure 3 Configurational comparison of CPP stereoisomers
AUTHOR CONTRIBUTION
The biochemical studies described in the present study were carried out by Dana Morrone,
with assistance from Matthew Mead and Luke Lowry. NMR analysis of the resulting novel
diterpenes were performed by Matthew Hillwig and Bruce Fulton. The manuscript was
prepared by Reuben Peters and Dana Morrone.
ACKNOWLEDGEMENTS
The authors thank Professor Robert M. Coates (University of Illinois, Urbana, IL, U.S.A.)
for critically reading the manuscript, and Dr Xiaoming Chen (Iowa State University) for
preparation of the rice tissues analysed in the present study.
FUNDING
This work was supported by the National Institutes of Health [grant number GM076324
(to R.J.P.)].
Figure 4 Comparison of active-site conformations of the alternative CPP
stereoisomers indicated by catalysed product outcome for (A) OsKSL4 and
(B) OsKSL11
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Received 8 September 2010/24 January 2011; accepted 16 February 2011
Published as BJ Immediate Publication 16 February 2011, doi:10.1042/BJ20101429
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The Authors Journal compilation 2011 Biochemical Society