Non-natural cinnamic acid derivatives as substrates of cinnamate 4-hydroxylase

Article abstract of DOI:10.1016/j.phytochem.2006.10.018

Cinnamate 4-hydroxylase (C4H), a monooxygenase in the plant phenylpropanoid pathway, was assayed for its ability to hydroxylate 29 substrate analogues. Nine of the tested analogues with various aromatic side chains, including 3-coumaric acid, were metabolized by C4H. Seven products from these reactive analogues were characterized using LC/MS, ¹H NMR and ¹³C NMR spectroscopic analysis. For example, caffeic acid was the product of 3-coumaric acid. The products 4-hydroxy-2-chlorocinnamic acid and 4-hydroxy-2-ethoxycinnamic acid are novel compounds that have not been previously reported. The kinetic parameters of C4H towards these analogues were determined.

Full text of DOI:10.1016/j.phytochem.2006.10.018

PHYTOCHEMISTRY
Phytochemistry 68 (2007) 306–311
www.elsevier.com/locate/phytochem
Non-natural cinnamic acid derivatives as substrates of cinnamate
4-hydroxylase
*
Hao Chen, Hanxiao Jiang, John A. Morgan
School of Chemical Engineering, Purdue University, 1055 FRNY Hall, West Lafayette, IN 47907-1283, USA
Received 9 June 2005; received in revised form 19 September 2006
Available online 1 December 2006
Abstract
Cinnamate 4-hydroxylase (C4H), a monooxygenase in the plant phenylpropanoid pathway, was assayed for its ability to hydroxylate
29 substrate analogues. Nine of the tested analogues with various aromatic side chains, including 3-coumaric acid, were metabolized by
1
C4H. Seven products from these reactive analogues were characterized using LC/MS, H NMR and 13C NMR spectroscopic analysis.
For example, caffeic acid was the product of 3-coumaric acid. The products 4-hydroxy-2-chlorocinnamic acid and 4-hydroxy-2-ethoxy-
cinnamic acid are novel compounds that have not been previously reported. The kinetic parameters of C4H towards these analogues
were determined.
ꢀ 2006 Published by Elsevier Ltd.
Keywords: Arabidopsis thaliana; Cruciferae; Saccharomyces cerevisiae; Cytochrome P450; Phenylpropanoids; Coumaric acid
1. Introduction
than two aromatic rings (Schoch et al., 2003). The metab-
olism of unnatural substrates by C4H provides the possibil-
ity to synthesize novel products from phenylpropanoid
pathway, if other enzymes in this pathway can further
metabolize those products. Also, since there is no crystal
structure currently available for C4H, the investigation of
alternative substrates provides insight into the binding
and reaction mechanism of C4H (Schoch et al., 2003).
In this study, we tested the ability of recombinant C4H
from Arabidopsis thaliana (CYP73A5) towards 29 ana-
logues of trans-cinnamic acid (1). Several analogues are
hydroxylated by C4H, and we characterized the structures
of the products and the kinetic parameters of the reactions.
Cinnamate 4-hydroxylase (C4H; EC 1.14.13.11) is a
cytochrome P450 in the plant phenylpropanoid pathway,
which leads to many important secondary metabolites
such as lignins, coumarins, and flavonoids (Humphreys
and Chapple, 2002; Weisshaar and Jenkins, 1998). These
compounds are essential to form structural components,
pigments, antioxidants, signaling molecules, UV-protec-
tive compounds, antibiotics and anti-insect compounds
in plants (Morant et al., 2003; Taylor and Grotewold,
2005; Weisshaar and Jenkins, 1998). Together with the
NADPH-P450 reductase and the cofactor NADPH,
C4H catalyzes the para-hydroxylation of trans-cinnamic
acid 1 to p-coumarate 2 (Fig. 1).
In addition to its natural substrate, C4H has the ability
to metabolize some substrate analogues, including xenobi-
otics (Schalk et al., 1997a,b; Urban et al., 1994). These ana-
logues are derivatives of cinnamate (1) with a planar
structure, negatively charged side chain and a size of less
2. Results and discussion
2.1. Alternative substrates of C4H
Based on structural similarity to cinnamate 1, we first
tested the reactivity of 15 analogues of trans-cinnamic acid
1 with different side chains on the aromatic ring in place of
acrylic acid, e.g., eugenol and phenylacetic acid, but none
*
Corresponding author. Tel.: +1 765 494 4088; fax: +1 765 494 0805.
E-mail address: jamorgan@purdue.edu (J.A. Morgan).
0031-9422/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.phytochem.2006.10.018

H. Chen et al. / Phytochemistry 68 (2007) 306–311
307
This result shows that the substrate recognition sites of
C4H accept many cinnamic acid analogues with R1-substi-
tutions of various size and polarity. This finding is consis-
tent with previous reports of C4H activity against
naphthalene derivatives, whose R1-substituted analogues
were similar in size (Pierrel et al., 1994; Schalk et al.,
1997a,b). However, compounds 8–10 with substitutions
at R2 were also metabolized. In general, steric effects and
hydrophobic interactions are keys to the specificity of
P450s (De Voss et al., 1997). It is relatively difficult to
deduce any influence on reactivity caused by the electronic
character of the analogues with substitution on R1, since
both electron withdrawing (F, Cl, NO2) and electron
donating (methyl, methoxy, ethoxy) substitutions were
hydroxylated.
Fig. 1. Reaction catalyzed by C4H in plants.
disappeared from solution in the in vivo feeding study with
recombinant C4H (data not shown). Next we similarly
tested the reactivity of 14 analogues with substitution at
R1 or R2 or both positions (Fig. 2). Compounds 3–7 were
not metabolized by the WAT11 host expressing C4H
enzyme. However, we can not rule out the possibility that
the absence of reactivity could be due to lack of uptake
of specific analogues. All analogues that were hydroxylated
are R1-substituents except 3-fluorocinnamic acid 8, 3-
methylcinnamic acid 9, and 3-hydroxycinnamic acid 10.
No formation of products was observed from analogues
fed to the control yeast strain lacking C4H.
2.2. Structures of products from substrate analogues
From a comparison of HPLC retention time, LC/MS
molecular mass and UV-spectra between the product
derived from 3-hydroxycinnamic acid 10 and a caffeic acid
17 standard, we confirmed the product is caffeic acid 17.
The LC/MS results for the products from compounds 8–
16 are consistent with the predicted molecular weights of
the hydroxylated substrate analogues.
The 1H NMR and 13C NMR data, UV spectra, and HR-
ESI-MS data for the metabolized products of substrates
10–15 are listed in Tables 1 and 2, respectively. Based on
the analysis of proton couplings, we propose that the
new hydroxyl group is always at the 4-position. The struc-
tures of compounds 18–22 are 2-fluoro-4-hydroxycinnamic
acid, 2-chloro-4-hydroxycinnamic acid, 2-methyl-4-
hydroxycinnamic acid, 2-methoxy-4-hydroxycinnamic
acid, and 2-ethoxy-4-hydroxycinnamic acid, respectively
1
1
(Fig. 2). Based on the analysis of H, 13C, H–1H COSY,
1H–13C HMQC, and H–13C HMBC spectra, we assigned
1
the positions of protons and carbons for 18–22 (Tables 1
and 2). The structure of compound 23 is proposed as 2-
nitro-4-hydroxycinnamic acid (Tables 1 and 2). Based on
the analysis of long range coupling of 1H and 13C, the
hydroxyl group in compound 23 is likely para- to the
acrylic acid side chain. Due to the low activity of C4H
against substrates 8 and 9, the products from these two
analogues were not available in sufficient quantity for
NMR experiments.
To the best of our knowledge, this is the first time these
analogues have been shown to be metabolized by C4H. The
products 4-hydroxy-2-chlorocinnamic acid 19 and 4-
hydroxy-2-ethoxycinnamic acid 22 are novel compounds
that have not been previously reported. The products 18,
20, 21, and 23 have been reported as intermediates for fine
chemicals syntheses in patents or the chemical literature
(Kaku et al., 2004; Bloom, 1959; Oonuma et al., 1993;
Khanna, 1983). Compound 21 was tentatively identified
in Hemarthria altissima (Tang and Young, 1982). Caffeic
acid 17, the product of compound 10, is an antioxidant
found in various plants (Yamanaka et al., 1997).
Fig. 2. Substrate analogues and products of C4H. Substrate analogues
(1 mM) were incubated at 30 ꢁC for 2 h with 10 mL of induced WAT11
cells expressing C4H activity. Substrates 3–9 were identified as non-
reactive if no decrease in concentration from HPLC chromatograms was
observed.

308
H. Chen et al. / Phytochemistry 68 (2007) 306–311
Table 1
1H (500 MHz, CD3OD) NMR data [d (ppm), m, J (Hz)]a, UV spectra [kmax (MeOH), nm], and HR-ESI-MS data for the products of C4H substrate
analogues
Substitution
Position
F
18
Cl
19
CH3
20
OCH3
21
OCH2CH3
22
NO2
23
3
5
6
20
30
R1
6.51 dd (12.5, 2.5)
6.61 dd (8.5, 2.5)
7.46 t (8.5)
6.34 d (16.0)
7.66 d (16.0)
6.82 d (2.5)
6.72 dd (8.5, 2.5)
7.58 d (8.5)
6.29 d (16.0)
7.96 d (16.0)
6.57
6.56
7.40 d (9.0)
6.24 d (15.5)
7.77 d (15.5)
2.28 s
6.40 d (2.0)
6.36 dd (8.5, 2.0)
7.36 d (8.5)
6.30 d (16.0)
7.84 d (16.0)
3.81 s
6.38 d (2.5)
6.35 dd (8.5, 2.5)
7.35 d (8.5)
6.33 d (16.0)
7.84 d (16.0)
4.02 q (7.0)
1.42 t (7.0)
284, 321
7.31 d (2.5)
7.07 dd (8.5, 2.5)
7.66 d (8.5)
6.32 d (16.0)
7.87 d (16.0)
kmax(nm)
289, 305
182.0392
182.0379
296
198.0094
198.0084
315
178.0634
178.0630
287, 323
194.0583
194.0579
256, 289
209.0334
209.0324
HR-ESI-MS m/z
Calc.b
208.0752
208.0736
a
Assignments were based on COSY, HMQC, and HMBC experiments.
Calculated for the formula C9H7O3R, R = substitutions.
b
Table 2
Humphreys and Chapple, 2004; Pierrel et al., 1994; Urban
et al., 1997, 1994).
13C (125 MHz, CD3OD) NMR data of the products from C4H reaction
with analoguesa
Not surprisingly, C4H has the highest catalytic efficiency
(kcat/Km) for the metabolism of trans-cinnamic acid 1. The
Km values for 2-fluorocinnamic acid 10, 2-chlorocinnamic
acid 11 and 2-ethoxycinnamic acid 15 are also close to that
of Km for 1. Of all the analogues, the metabolism of 10 by
C4H has the highest efficiency (70.3 minꢀ1 lMꢀ1) and the
second highest turnover number (164.6 15.1 minꢀ1).
The Km and catalytic efficiency for the metabolism of 10
and 11 were comparable to another reported substrate ana-
logue of C4H, 2-naphthoic acid (Schalk et al., 1997a).
Position
18b
19
20
21
22
23
1
2
3
4
5
6
10
20
30
R1
114.8 d (12)
163.8 d (273)
103.9 d (24)
162.8 d (10)
113.3
131.2 d (5)
172.0
118.3
124.7
136.9
116.2
161.6
117.4
130.0
170.5
118.4
141.7
124.5
139.6
116.8
159.3
113.3
127.8
170.4
115.6
142.0
18.4
114.6
160.2
98.4
161.3
107.6
129.9
170.3
114.0
140.7
54.5
116.1
160.9
100.7
162.6
109.0
131.5
171.8
115.4
142.4
65.1
121.6
151.2
112.0
161.0
121.6
131.2
170.4
122.0
140.5
138.9
15.0
a
Assignments were based on COSY, HSQC, and HMBC experiments.
Carbons 1, 2, 3, 4, and 6 of the compound 18 are split by 19F. Numbers
3. Concluding remarks
b
in the brackets indicate the values of JCF (Hz).
Most previous reports of active C4H substrate analogues
are mimics of 2-naphthoic acid with two heterocyclic rings
(Pierrel et al., 1994; Schalk et al., 1997a,b). With the excep-
tion of 2-naphthoic acid, C4H had a very low catalytic effi-
ciency (less than 0.8 minꢀ1 lMꢀ1) towards the other
documented analogues (Pierrel et al., 1994; Schalk et al.,
1997a,b). Also, with side chains other than acrylic acid,
the p-hydroxylase function of C4H towards those reported
analogues was not well maintained (Pierrel et al., 1994).
Similarly, from our study, C4H did not react with ana-
logues without acrylic acid substitution. In general, the cat-
alytic efficiency of C4H towards R1 substituents decreased
when the size of substituents increased. However, it is diffi-
cult to explain the variation of kinetic parameters because
the trend does not directly correlate either with the size or
the electronic effects of the analogues.
2.3. Determination of kinetic parameters
The kinetic parameters of C4H towards these analogues
were determined through in vitro enzyme assays and the
results are reported in Table 3. Both the Km and the turnover
number of C4H towards cinnamic acid 1 were found to be
consistent with the values reported in the literature, varying
from 0.7 lM to 8.9 lM, and 28 minꢀ1 to 129.1 minꢀ1
respectively (Gravot et al., 2004; Hubner et al., 2003;
,
Table 3
Kinetic parameters of C4H with substrate analogues
R1
R2
Km (lM)
kcat (minꢀ1
)
kcat/Km
(minꢀ1 lMꢀ1
)
1
11
12
10
14
16
15
13
H
F
Cl
H
H
H
H
0.5 0.1 102.9 4.9
2.3 0.3 164.6 15.1
197.9
70.3
29.5
17.8
15.3
10.8
3.4
2.2 0.4
65.8 8.6
4. Experimental
OH 13.1 2.3 232.0 24.2
CH3
H
H
H
H
10.0 2.4 153.4 2.1
4.1. General
CH3CH2O
CH3O
NO2
6.5 1.7
18.4 1.4
21.5 2.4
70.2 15.8
62.1 34.2
27.5 7.1
4.1.1. Chemicals
1.3
Isoeugenol and 2-chlorocinnamic acid were purchased
from ACROS Organics (Geel, Belgium). 3-Methoxycin-
namic acid, 2-methyl cinnamic acid, eugenol, 2-methoxycin-
A reaction mixture containing 5–30 lL C4H microsomes, 0.5–40 lM
substrate, and 1 mM NADPH, in a final volume of 500 lL was incubated
at 30 ꢁC for 3–10 min. Further details are described in Section 4.

H. Chen et al. / Phytochemistry 68 (2007) 306–311
309
namic acid, and 4-hydroxy-3-methoxy phenyl acetone were
purchased from Alfa Aesar (Ward Hill, MA). Benzene and
toluene were from Mallinckrodt Baker, Inc. (Paris, Ken-
tucky). 4-Hydroxy-3-methoxyphenyl pyruvic acid was pur-
chased from TCI America (Portland, OR). Yeast nitrogen
base without amino acids and ammonium sulfate (YNB-
AA/AS) was purchased from Becton Dickinson (Sparks,
MD). 3-Nitrocinnamic acid, cinnamyl acetate, methyl trans-
cinnamate, 2-fluorocinnamic acid, trans-2,3-dimethoxy-
cinnamic acid, 3-fluorocinnamic acid, trans-2-methoxy-4-
(2-nitrovinyl) phenol, 3-methylcinnamic acid, benzoic acid,
2-ethoxycinnamic acid, 3-cholorocinnamic acid, homova-
nillic acid, 2-hydroxy cinnamic acid, 3-hydroxycinnamic
acid, 2-nitrocinnamic acid, phenylacetic acid, tropic acid,
mandelic acid, hippuric acid, glucose, galactose, casein
enzymatic hydrolysate (EHC), L-tryptophan, methanol,
acetonitrile, dimethyl sulfoxide (DMSO), trans-cinnamic
acid and p-coumaric acid were of the highest purity and pur-
chased from Sigma–Aldrich Co. (St. Louis, MO).
terated methanol (CD3OD) as the solvent and tetrameth-
ylsilane (TMS) as an internal standard. NMR data were
reported with d (ppm) values, and referenced to the solvent.
UV spectra were obtained on a DUꢂ Series 500 spectro-
photometer (Beckman Instruments, Inc., CA) in MeOH.
4.2. Yeast strain, culture medium and growth conditions
4.2.1. Strain and plasmids
A Saccharomyces cerevisiae strain, WAT11, derived
from the W303-B strain (MAT a; ade2-1; his3-11, -15;
leu2-3, -112; ura3-1; canR; cyr+) with an Arabidopsis thali-
ana NADPH-cytochrome P450 reductase gene integrated
into chromosome was used as the host strain (Pompon
et al., 1996). The S. cerevisiae expression vector pYeDP60
driven by a GAL10-CYC1 hybrid promoter was constructed
by Pompon et al. (1996). The pYeDP60 vector containing a
C4H gene from Arabidopsis thaliana (CYP73A5) was con-
structed by Humphreys and Chapple (2004). The WAT11
containing pYeDP60-C4H vector was used as host strain
and the WAT11 containing an empty pYeDP60 vector
was used as control strain to test for biotransformation of
the substrates by the yeast host strain.
4.1.2. HPLC method
Analysis of the in vitro reaction was performed by HPLC
using an Agilent (Palo Alto, CA) SB-C18 column (4.6 ·
75 mm) at 30 ꢁC with a gradient elution method. The flow
rate was 0.9 mL/min and the injection volume was 10 lL.
Solvent A was 1.5% (v/v) AcOH in H2O and solvent B
was CH3CN. Initially, solvent B was maintained at 5%
for 4 min, increased to 45% over 8.5 min, and held for
1 min before returning to 5% solvent B. The UV absorbance
at kmax (Table 2) for respective product was recorded.
4.2.2. Culture media
The medium composition was based on a previously
optimized medium for ferulate 5-hydroxylase expression
in the WAT11 strain (modified synthetic medium SGI)
(Jiang and Morgan, 2004). The composition of the growth
medium is 20 g/L glucose, 5 g/L casein enzyme hydroly-
sate, 40 mg/L L-tryptophan, and 3.4 g/L YNB-AA/AS.
The induction medium was optimized for C4H expression
(Chen and Morgan, 2006) and the composition is 3.3 g/L
glucose, 16.7 g/L galactose, 7.4 g/L casein enzyme hydroly-
sate, 40 mg/L L-tryptophan and 3.4 g/L YNB-AA/AS.
4.1.3. Mass spectrometry
Electrospray ionization (ESI) analyses were carried out
on a Waters Micromass Q-TOF system (Waters, Milford,
MA) after HPLC on
a
Waters C18 column
(2.1 · 150 mm) with a flow rate of 0.2 mL/min by a gradi-
ent elution method. Initially, solvent B was maintained at
5% for 8 min, increased to 45% over 17 min, and held for
5 min before returning to 5% solvent B. The mass spec-
trometer scan range was from 50 to 600 m/z and detection
was in the negative ion mode. Flow injection analyses were
carried out on an Agilent MSD TOF mass spectrometer
with an ESI source. The flow rate was 0.5 mL/min with
an eluent H2O–CH3CN (1:1). The acquisition range was
from 40 to 1000 m/z and detection was in the negative
ion mode. The products from compounds 8 and 9 were
analyzed with the same MS systems after HPLC on the
Agilent system with the same gradient elution profile as
previously described. The flow rate was adjusted to
0.8 mL/min, and the sample injection volume was 15 ll.
The content of solvent A was changed to 0.1% AcOH in
H2O and solvent B was CH3CN with 0.1% AcOH. The
spectra were collected in negative ion mode.
4.2.3. Cell cultivation procedures
A single yeast colony from a plate containing modified
SGI medium was transferred to 25 mL of modified SGI
medium in a 125 mL flask and cultivated at 30 ꢁC with
shaking at 300 rpm for 24–28 h until the OD600 reached
1.0–2.0. The cells were then subcultured to a 1 L flask with
250 mL induction medium and diluted to an initial OD600
of 0.02. The cell culture was induced at 30 ꢁC for 20 h with
a shaking speed of 300 rpm.
4.3. In vivo reaction for analogue screening and product
preparation
For the substrate analogue activity assays, 1 mM ana-
logue was added to 10 mL of induced cells expressing
C4H at 30 ꢁC for 2 h. Samples (500 lL) of the reaction mix-
ture were removed and mixed with and equal volume of
MeOH immediately to terminate the reaction. The mixture
was centrifuged at 18,000g and the supernatant was ana-
lyzed by HPLC. A decrease of substrate and appearance
of a new peak with earlier retention time were evidence of
4.1.4. NMR analyses
1H and 13C NMR spectroscopic data were obtained on
Bruker DRX 500 NMR spectrometer (500 MHz) with deu-

310
H. Chen et al. / Phytochemistry 68 (2007) 306–311
reaction. For preparation of product for structural identifi-
cation, substrate analogues were added at a final concentra-
tion of 1 mM–250 mL of induced yeast cells. The reactions
was terminated after 26–33 h.
was from a linear plot of reaction velocity against substrate
concentration.
4.7. Identification of enzyme reaction products
4.4. Extraction and purification
Compound characterization data for products 18–23 are
described in Tables 1 and 2 (1H, 13C and HRMS data).
After reaction, the cell culture was centrifuged at 5000
rpm for 10 min at 4 ꢁC. The supernatant was acidified by
AcOH (5:1 v/v) and the mixture was extracted twice with
equal volumes of EtOAc. The solvent was removed in
vacuo and the sample was injected multiple times on an
Agilent SB-C18 column (9.4 · 100 mm). The new product
peaks were separated by an isocratic elution method. Sol-
vent A was 1.5% (v/v) AcOH in H2O and solvent B was
100% CH3CN. Solvents A and B were mixed as 15:85 (v/
v) at a flow rate of 4 mL/min. Product peaks were collected
and the solvent was evaporated to yield dry product.
Acknowledgements
The work was funded by the Shreve trust fund from the
School of Chemical Engineering, Purdue University. The
authors thank Prof. C. Chapple (Purdue University) for
his donation of the pYeDP60-C4H plasmid in the
WAT11 host. We thank the Purdue Interdepartmental
NMR Facility for the NMR spectroscopy. We would also
like to thank Bruce Cooper from the Bindley Bioscience
Center and the campus wide mass spectrometry facility
for the LC/MS analyses. MS analysis was conducted at
the Purdue University Metabolic Profiling Facility, sup-
ported by the National Science Foundation under Grant
DBI-0421102.
4.5. Microsome preparation and C4H quantification
After induction, microsomes were prepared essentially
as described by Urban et al. (1994). The yeast cells were
washed and resuspended in a high-salt Tris buffer (pH
7.6, 50 mM Tris–HCl, 20% glycerol, 4 mM EDTA and
150 mM M NaCl). Zirconia beads were added to the resus-
pended cells and the mixture was vortexed vigorously.
After centrifugation, the pellets were discarded and PEG
3350 was slowly added to the supernatant to a concentra-
tion of 0.1 g/mL, with stirring at 4 ꢁC for 15 min before
it was centrifuged at 10,000g for 10 min. The pellets were
resuspended in PIPES buffer (pH 7.0, 50 mM PIPES,
20% glycerol, 4 mM EDTA) using a Dounce homogenizer
to create a uniform microsome suspension. Samples were
divided into 500 lL aliquots and immediately stored at
ꢀ80 ꢁC.
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4.6. In vitro enzyme kinetics assay
A series of concentrations of substrates from 0.5 lM to
40 lM were incubated at 30 ꢁC with 1 mM NADPH. Reac-
tions were initiated with aliquots of yeast microsomes (5–
30 lL) in a total volume of 500 lL. Samples (150 lL) of
the reaction mixture were taken at different time intervals
and mixed with 30 lL acetic acid immediately to terminate
reaction. The mixture was centrifuged at 14,000 rpm and
the supernatant was analyzed by HPLC. Substrate disap-
pearance was correlated with product peak area to achieve
a standard curve of product concentration. The Vmax was
calculated from the Lineweaver–Burk plot, and the Km

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