The efficient and selective catalytic oxidation of para-substituted cinnamic acid derivatives by the cytochrome P450 monooxygenase, CYP199A4

Article abstract of DOI:10.1039/c6ra11025h

The cytochrome P450 enzyme, CYP199A4, demethylated 4-methoxybenzoic acid, but not 4-methoxyphenylacetic acid, with high product formation activity. The oxidative demethylation of 3-(4-methoxyphenyl)propionic acid was 8-fold more active than 4-methoxyphenylacetic acid and 4-methoxycinnamic acid was efficiently oxidised at a product formation rate of 180 nmol nmol-P450^-1 min^-1. Accordingly the oxidation of cinnamic acid derivatives was investigated in order to determine the potential of CYP199A4 to act as a biocatalyst for this important class of biological molecules. 4-Methoxy- and 4-methyl-cinnamic acids bound tightly to CYP199A4 and were better substrates for CYP199A4 than cinnamic acid itself. The oxidations of both 4-methoxy- and 4-methyl-cinnamic acids was 100% selective for attack at the para substituent. Certain dimethoxy substituted cinnamic acids were demethylated more efficiently than 4-methoxycinnamic acid and retained the selectivity for the para-methoxy substituent. Only very low product turnover was observed with 3,5-dimethoxycinnamic acid. 4-Isopropylcinnamic acid was hydroxylated and desaturated by CYP199A4 at the isopropyl group. Cinnamic acids with a para-substituted alkyl- and alkyloxy-cinnamic acid framework were a good fit for the active site of the CYP199A4 enzyme and as a consequence were efficiently and selectively oxidised. Whole-cell oxidations resulted in high yields of product and CYP199A4 could be developed for applications in the biocatalytic oxidation of cinnamic acid derivatives and related phenylpropanoids.

Full text of DOI:10.1039/c6ra11025h

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Page 1 of 34
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The efficient and selective catalytic oxidation of para-substituted cinnamic acid
derivatives by the cytochrome P450 monooxygenase, CYP199A4
a
b
a*
Rebecca R. Chao , James J. De Voss and Stephen G. Bell
a
Department of Chemistry, University of Adelaide, SA 5005, Australia. Fax: +61 8 8303 4380;
email: stephen.bell@adelaide.edu.au
b
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Qld, 4072,
Australia.

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Abstract
The cytochrome P450 enzyme, CYP199A4, demethylated 4ꢀmethoxybenzoic acid, but not 4ꢀ
methoxyphenylacetic acid, with high product formation activity. The oxidative demethylation of 3ꢀ
(
4ꢀmethoxyphenyl)propionic acid was 8ꢀfold more active than 4ꢀmethoxyphenylacetic acid and 4ꢀ
methoxycinnamic acid was efficiently oxidised at a product formation rate of 180 nmol nmol
−
1
−1
p450 min . Accordingly the oxidation of cinnamic acid derivatives was investigated in order to
determine the potential of CYP199A4 to act as a biocatalyst for this important class of biological
molecules. 4ꢀMethoxyꢀ and 4ꢀmethylꢀcinnamic acids bound tightly to CYP199A4 and were better
substrates for CYP199A4 than cinnamic acid itself. The oxidations of both 4ꢀmethoxyꢀ and 4ꢀ
methylꢀcinnamic acids was 100% selective for attack at the para substituent. Certain dimethoxy
substituted cinnamic acids were demethylated more efficiently than 4ꢀmethoxycinnamic acid and
retained the selectivity for the paraꢀmethoxy substituent. Only very low product turnover was
observed with 3,5ꢀdimethoxycinnamic acid. 4ꢀIsopropylcinnamic acid was hydroxylated and
desaturated by CYP199A4 at the isopropyl group. Cinnamic acids with a paraꢀsubstituted alkylꢀ
and alkyloxyꢀcinnamic acid framework were a good fit for the active site of the CYP199A4 enzyme
and as a consequence were efficiently and selectively oxidised. Wholeꢀcell oxidations resulted in
high yields of product and CYP199A4 could be developed for applications in the biocatalytic
oxidation of cinnamic acid derivatives and related phenylpropanoids.
2

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Introduction
The hemeꢀdependent cytochrome P450 (CYP) monooxygenase enzymes primarily catalyse the
insertion of an oxygen atom derived from dioxygen into the carbonꢀhydrogen bonds of organic
1
ꢀ3
molecules. CYP199A4, from Rhodopseudomonas palustris strain HaA2, is able to selectively
4, 5
demethylate 4ꢀmethoxybenzoic acid.
It is also able to hydroxylate and desaturate paraꢀ
substituted alkyl benzoic acids such as 4ꢀethylꢀ and 4ꢀisopropylꢀbenzoic acids to form a mixture of
4, 6
products. These activities are supported by a class I electron transfer chain consisting of a [2Feꢀ
2
S] ferredoxin (HaPux) and a flavinꢀdependent ferredoxin reductase (HaPuR) which mediates heme
4
reduction by NADH. When CYP199A4 was paired with these electron transfer partners, the
activities of oxidative demethylation, hydroxylation and desaturation of paraꢀsubstituted benzoic
4
, 7, 8
acids are high.
Mechanistically, C−H bond hydroxylation occurs via hydrogen abstraction by Compound I,
2
, 9
followed by oxygen rebound to give the hydroxylated organic.
Oꢀdemethylation of 4ꢀ
methoxybenzoic acid by CYP199A4 occurs via the same pathway, initially yielding a hemiacetal
2
, 6, 10, 11
which spontaneously decomposes to yield 4ꢀhydroxybenzoic acid and formaldehyde.
The
alkene desaturation product is hypothesised to arise from initial hydrogen atom abstraction followed
by either a second hydrogen atom abstraction from an adjacent carbon or oneꢀelectron oxidation by
Compound II. The latter pathway generates a carbocation which then loses a proton to form the
2
, 6
alkene.
A detailed knowledge of the types of substrates that can bind in the enzyme active site and
the interactions responsible for binding is of paramount importance in achieving the activity and
1
2ꢀ15
turnover numbers required for synthetic applications.
The stable, soluble nature and high
activity of the CYP199A4 enzyme means it is well suited for larger scale processes involving C–H
4, 6, 16ꢀ18,19
bond oxidation such as hydroxylation and oxidative demethylation.
Altering the benzene
ring, the carboxylate group or the removal of the methoxy substituent of 4ꢀmethoxybenzoic acid all
3

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4
, 6, 20, 21
reduced the substrate binding and monooxygenase activity of CYP199A4.
Replacement of
the methoxy group with alternate substituents is tolerated as are small additional functional groups
6
on the benzene ring. We have shown that CYP199A4 can bind and Oꢀdemethylate 3,4ꢀ
dimethoxybenzoic acid with total regioselectivity at the 4ꢀmethoxy group and demethenylate 3,4ꢀ
4
, 20
methylenedioxybenzoic acid.
Others have reported that the CYP199A subfamily member,
CYP199A2 from R. palustris CGA009, which shares > 85% sequence identity with CYP199A4,
can catalyse the oxidation of larger substrates such as 2ꢀnaphthoic acid, indoleꢀ6ꢀcarboxylic acid
4
, 16ꢀ18, 22ꢀ24
and 3ꢀhydroxyꢀ and 4ꢀhydroxyꢀcinnamic acids (mꢀ and pꢀcoumaric acids, respectively).
Mutant forms of CYP199A2 have been shown to enhance the biocatalytic properties of CYP199A2,
allowing the wholeꢀcell oxidation of cinnamic acid, benzyl alcohols and phenols using an E. coli
19
system. However, detailed in vitro analysis of the substrate binding interactions and enzyme
activity with these substrates has not been reported.
Several crystal structures of substrateꢀfree and substrateꢀbound forms of CYP199A4 and
5
, 6, 10
CYP199A2 have been determined.
In the substrateꢀbound forms (PDB codes; 4DO1, 4EGM
and 4EGN), the substituent para to the carboxylate is oriented towards the hemeꢀiron which is
consistent with exclusive attack at this position. The carboxylate group of the substrate interacts
with the polar side chains of Arg92, Ser95, Ser244 and, via a bridging water molecule, Arg243
(
Fig. 1). In addition, hydrophobic interactions with the benzene ring help to position the substrate.
The crystal structures of CYP199A4 in complex with the larger indoleꢀ6ꢀcarboxylic acid and 2ꢀ
naphthoic acid substrates have also been solved (PDB codes; 4EGO and 4EGP). In the indoleꢀ6ꢀ
carboxylic acidꢀbound structure, the benzene rings, the carboxylate groups and the bridging water
5
molecules are at virtually identical locations to those observed with 4ꢀmethoxybenzoic acid.
Although 2ꢀnaphthoic acid is similar in size to indoleꢀ6ꢀcarboxylic acid, its crystallographically
observed binding mode is significantly different. The carboxylate group is located ca. 1.9 Å closer
to the Nꢀterminal end of the I helix. The water molecule found in the 4ꢀmethoxybenzoic acidꢀbound
4

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form, which interacts with the substrate and Arg243, is displaced and the carboxylate group of 2ꢀ
5
naphthoic acid interacts directly with Arg243. The hydrogen bonds between the carboxylate
oxygen atom and the side chains of Ser95 and Ser244 are maintained. Together, these structures
5
show that CYP199A4 is able to accommodate larger planar and acidic substrates. By way of
contrast, it has been shown that small divergences from 4ꢀmethoxybenzoic acid structure can result
in a dramatic reduction in the enzyme activity and in the affinity for the substrate. For example, the
activity of CYP199A4 with 4ꢀmethoxyphenylacetic acid, which is non planar, is 240ꢀfold lower
than that with 4ꢀmethoxybenzoic acid and there is a three orders of magnitude reduction in the
2
1
substrate binding affinity. This highlights the specificity of the hydrophilic interactions between
the CYP199A4 enzyme and the aromatic acid group of the substrate.
To further understand these interactions, we compare the binding and kinetic properties of
CYP199A4 with selected cinnamic acid derivatives (Fig. 2). These cinnamic acids belong to the
phenylpropanoid family of natural products and have a variety of biological activities and
25, 26
functions.
Cinnamic acids have an unsaturated planar carboxylate containing side chain and
will be compared with related nonꢀplanar substrates. The requirement of a paraꢀsubstituent in these
larger substrate and the effect of other substituents at the other locations of the benzene ring will
also be investigated. The aim is to provide a better understanding of the structural features required
for tight binding to this family of CYP enzymes. In addition the size and shape of substrates that
can be efficiently oxidised by CYP199A4 will be determined which will facilitate its use as a
biocatalyst on a synthetically useful scale.
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Results
Substrate binding studies on the CYP199A4 system
It has been reported that 4ꢀhydroxycinnamic acid and 3ꢀhydroxycinnamic acids are substrates for
CYP199A2 from R. palustris CGA009 with the wholeꢀcell oxidation of both giving rise to 3,4ꢀ
2
3, 24
dihydroxycinnamic acid.
The amino acid sequences and substrate binding profiles of the
4
CYP199A2 and CYP199A4 enzymes are very similar. The binding of both 3ꢀ and 4ꢀ
hydroxycinnamic acids and the parent compound, cinnamic acid to CYP199A4, were studied. The
addition of all of these substrates to CYP199A4 resulted in a spin state shift to the highꢀspin form of
≤
5% (Table 1 and Fig. S1). 4ꢀHydroxycinnamic acid induced no observable spin state shift upon
addition to CYP199A4. Due to the negligible shift in the spin state, an accurate determination of the
dissociation constant was not possible for any of these substrates.
We have previously shown that a substituent para to the carboxylate moiety of benzoic acid
6, 20,
was important for binding and efficient catalytic activity with both CYP199A2 and CYP199A4
2
2
and 4ꢀmethoxybenzoic acid was the optimal substrate for both with CYP199A4 (≥95%, spin state
4
, 5, 10
shift and a K
d
of 0.28 ꢀM).
Accordingly, we tested the binding of 4ꢀmethoxycinnamic acid
with CYP199A4. This substrate induced a spin state shift of 70% on binding to CYP199A4 and the
dissociation constant was determined to be 3.6 ꢀM (Table 1 and Fig. 3 and 4). By way of contrast,
2
1
4
ꢀmethoxyphenylacetic acid binds poorly: spin state shift 5%; K
Methoxyphenyl)propionic acid induced a lower spin shift to the highꢀspin state (20%) and weaker
binding (K , 31 ꢀM ) with CYP199A4 than both 4ꢀmethoxybenzoic and 4ꢀmethoxycinnamic acids.
d
690 ꢀM.
3ꢀ(4ꢀ
d
However the magnitude of this shift and the binding affinity is higher than is observed with 4ꢀ
methoxyphenylacetic acid and cinnamic acid (Table 1, Fig. 3 and 4).
Due to the favourable results obtained with 4ꢀmethoxycinnamic acid, we tested a range of
substituted cinnamic acids with CYP199A4 to better determine the optimal size and shape of the
molecules which complement the substrate binding pocket (Fig. 2). Additional substitutions at the
6

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2
ꢀ and 3ꢀpositions of 4ꢀmethoxycinnamic acid tended to reduce the affinity for binding to
CYP199A4. 3ꢀHydroxyꢀ4ꢀmethoxyꢀ, 3,4ꢀdimethoxy, 2,4ꢀdimethoxyꢀ and 3,4ꢀmethylenedioxyꢀ
cinnamic acids all induced significantly lower spin state shifts upon addition to CYP199A4 and
resulted in weaker binding when compared to 4ꢀmethoxycinnamic acid (Table 1 and Fig. S1). 2,4ꢀ
Dimethoxycinnamic acid bound the most tightly to CYP199A4 of any of the disubstituted
substrates investigated despite possessing one of the lowest spin state shifts observed (Table 1).
Substrates containing methoxy groups at the 3ꢀ or 5ꢀpositions with a hydrogen atom or a hydroxyl
group at the 4ꢀposition such as 3,5ꢀdimethoxycinnamic acid, 3ꢀmethoxyꢀ4ꢀhydroxycinnamic acid
(
ferulic acid) and 3,5ꢀdimethoxyꢀ4ꢀhydroxycinnamic acid (sinapic acid) resulted in little observable
shift in the spin state upon binding to CYP199A4 (Table 1 and Fig. S1). Two trimethoxy
substituted cinnamic acids were tested with CYP199A4. Addition of neither 2,3,4ꢀ
trimethoxycinnamic nor 3,4,5ꢀtrimethoxycinnamic acids induced a significant spin state shift (Table
1
).
The effect of the size and type of the para substituent was also investigated with alkyl
substituted cinnamic acids. Upon binding to CYP199A4, 4ꢀmethylcinnamic acid induced a similar
spin state shift to 4ꢀmethoxycinnamic acid but the binding was 6ꢀfold weaker (Table 1 and Fig. 3
and 4). The increased size of the alkyl paraꢀsubstituent in 4ꢀisopropylcinnamic acid resulted in the
largest spin state shift observed, ≥ 95% highꢀspin and a comparable binding affinity to 4ꢀ
methoxycinnamic acid (Table 1 and Fig. S2). The trend in the CYP199A4 substrate binding affinity
for the alkyl and methoxy paraꢀsubstituted cinnamic acids mirrors that of equivalent paraꢀ
substituted benzoic acids (methoxy > isopropyl > methyl) despite the different trend in the spin
5
state shifts. On the whole, appropriately substituted cinnamic acids appear to be good target
substrates for oxidation by CYP199A4.
Activity and product formation assays
7

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The rate of NADH oxidation during the CYP199A4 catalysed oxidative demethylation of 4ꢀ
−1
−1
ꢀ1
methoxycinnamic acid was 560 nmol nmolꢀP450 min (henceforth abbreviated to min , Table 1
and Fig. 5). The product formation rate, which is the rate at which substrate is oxidised to product,
−1
−1 4
was inferior (180 min ) to that observed for the equivalent benzoic acid (1220 min ). This was
predominantly due to diminished coupling efficiency of the NADH reducing equivalents to product
6
formation (32% vs. 89ꢀ90%). The oxidation of 4ꢀmethoxycinnamic acid by CYP199A4 resulted in
the formation of a single product arising through demethylation of the methoxy group. This was
identified by HPLC coꢀelution of a 4ꢀhydroxycinnamic acid standard with the turnover (Fig. 6 and
Scheme 1). We note that two peaks were observed in the HPLC and GC analysis of both the
substrate and product controls of 4ꢀmethoxyꢀ and 4ꢀhydroxyꢀcinnamic acids. MS analysis indicated
that these likely arise from the E and Z isomers (Fig. 6 and S4) with the starting, stable E isomer
presumed to be present in excess in both instances. The NMR spectra of both substrate and product
samples showed the presence of predominantly the trans isomer (>99%). Product samples isolated
after wholeꢀcell oxidation and HPLC purification had coupling constants for the alkene hydrogens
consistent with a trans configuration (see supporting information). Overall isomerisation may be
2
7
occurring before or during the chromatographic analysis of the turnovers.
In accord with the reduced spin state shift and weaker binding of 3ꢀ(4′ꢀ
methoxyphenyl)propionic acid to CYP199A4, the rate of NADH oxidation was lower than that for
ꢀ1
4
ꢀmethoxycinnamic acid (98 min , Table 1 and Fig. 5). The coupling efficiency was higher than
that observed with 4ꢀmethoxycinnamic acid and 4ꢀmethoxyphenylacetic acid, resulting in a product
ꢀ1
formation rate of 43 min (Table 1). A single product was observed in the HPLC analysis of the
turnover which coꢀeluted with 3ꢀ(4′ꢀhydroxyphenyl)propionic acid (Scheme 1 and Fig. S3).
The addition of cinnamic acid and 3ꢀhydroxycinnamic acid to CYP199A4 resulted in no
appreciable increase in the rate of NADH oxidation above that observed in absence of substrate. 4ꢀ
Hydroxycinnamic acid induced a very small increase in NADH consumption (Table 1).
8

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Chromatographic analysis of the CYP199A4 catalysed oxidation of these two substrates revealed
little product formation (Fig. S3) but 3ꢀhydroxycinnamic acid did yield very low levels of 3,4ꢀ
dihydroxycinnamic acid (Fig. S3). HPLC analysis of the cinnamic acid turnover showed trace
amounts of 3ꢀ and 4ꢀhydroxycinnamic acids, though most if not all of the 3ꢀhydroxycinnamic acid
could potentially be accounted for from impurities in the substrate (Fig. S3).
Minimal increases in the rate of NADH oxidation above that of the background NADH
oxidase activity of the HaPux/HaPuR/CYP199A4 system, in the absence of substrate, were
observed with 3ꢀmethoxyꢀ4ꢀhydroxyꢀ, 3,5ꢀdimethoxyꢀ and 3,5ꢀdimethoxyꢀ4ꢀhydroxyꢀcinnamic
acids (Table 1). No product arising from substrate oxidation could be identified from CYP199A4
mediated oxidation of 3ꢀmethoxyꢀ4ꢀhydroxycinnamic acid or 3,5ꢀdimethoxyꢀ4ꢀhydroxycinnamic
acid, but very low levels of a single product were detected from 3,5ꢀdimethoxycinnamic acid (Fig.
S3). GCꢀMS analysis revealed a product with a mass consistent with 3ꢀhydroxyꢀ5ꢀ
methoxycinnamic acid (expected mass: 338.6, observed mass: 338.3; Fig. S4 and S5), the product
from a single oxidative demethylation.
Product formation from CYP199A4 catalysed oxidation of 3,4ꢀ(methylenedioxy)cinnamic
was 30ꢀand 50ꢀfold slower than that observed with 4ꢀmethoxyꢀ and 3,4ꢀdimethoxyꢀcinnamic acids
(
(
Table 1). The lower amount of product formed is mainly due to low coupling efficiency, 13%
Table 1). The sole product was identified as 3,4ꢀdihydroxycinnamic acid arising from
demethenylation (Fig. 6 and Scheme 1). The oxidation of 3,4ꢀdimethoxyꢀ, 2,4ꢀdimethoxyꢀ and 3ꢀ
hydroxyꢀ4ꢀmethoxyꢀcinnamic acids by CYP199A4 resulted solely in the demethylation of the
methoxy group at the 4ꢀposition and formation of a single product (Fig. S3 and S6). Despite the
lower spin state shifts and weaker binding to the enzyme compared to 4ꢀmethoxycinnamic acid, the
rate of NADH oxidation was high in all cases (Table 1). In fact, the levels of metabolite generated
exceeded that of 4ꢀmethoxycinnamic acid due to higher coupling efficiencies (59ꢀ75%) and resulted
ꢀ1
in product formation rates in excess of 200 min (Table 1).
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The turnover of 3,4,5ꢀtrimethoxycinnamic acid by CYP199A4 did not result in any NADH
oxidation activity above that of background NADH oxidase activity and no product formation was
observed. 2,3,4ꢀTrimethoxycinnamic was oxidised by CYP199A4 to generate a single product (Fig.
6
and Scheme 1). Although the NADH oxidation activity was low the coupling efficiency of this
ꢀ1
turnover was high resulting in a product formation rate of 24 min (Table 1). GCꢀMS analysis
indicated that the product arose from a single oxidative demethylation (substrate expected mass:
3
10.4, observed mass: 310.0; oxidative demethylation product; expected mass: 368.6, observed
mass: 368.0; Fig. S4 and S5). The product was assigned as 4ꢀhydroxyꢀ2,3ꢀdimethoxycinnamic acid
based on the exclusive attack on the paraꢀsubstituent reported so far for CYP199A4.
Substitution of the methoxy group with a methyl group resulted in a reduction in the NADH
ꢀ1
oxidation activity and product formation rate of the enzyme, 85 min (Table 1). However, the
coupling of the CYP199A4 turnover of 4ꢀmethylcinnamic acid was higher than that of 4ꢀ
methoxycinnamic acid (Table 1). As a result, the relative reduction in activity reported is not as
dramatic as that observed for the CYP199A4 catalysed turnover of 4ꢀmethylbenzoic acid compared
6
to 4ꢀmethoxybenzoic acid. A single product was observed in the CYP199A4 mediated oxidation of
4
ꢀmethylcinnamic acid (Fig. 6). This was generated in higher yield using a wholeꢀcell oxidation
system (vide infra), purified via semiꢀprep HPLC and characterised via NMR as 4ꢀ
hydroxymethylcinnamic acid (Scheme 1 and Fig. S6).
Despite the higher spin state shift of CYP199A4 with 4ꢀisopropylcinnamic acid, the
enzymeꢀcatalysed oxidation of this substrate was slower than that of 4ꢀmethylcinnamic acid (Table
1
). Four products were observed in the analysis of the in vitro turnovers by HPLC and GCꢀMS
(
Scheme 2 and Fig. S6). These products were also formed when using the wholeꢀcell oxidation
system, although in different proportions. The masses of the derivatised products indicated one
resulted from dehydrogenation, two others from hydroxylation and another from further oxidation
6
, 10, 28
of the alkene or an alcohol product (Fig. S5 and S6 and Table S2).
The products were
1
0

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generated using a wholeꢀcell oxidation system and three were isolated and purified by HPLC. They
were identified by NMR as 4ꢀ(1′ꢀhydroxyisopropyl)cinnamic acid, 4ꢀ(2′ꢀ
hydroxyisopropyl)cinnamic acid and 4ꢀ(1′,2′ꢀepoxyisopropyl)cinnamic acid (Scheme 1 and
Supporting information). The epoxide product presumably arises from further oxidation of 4ꢀ(propꢀ
1
′ꢀenꢀ2′ꢀyl)cinnamic acid, the desaturation product of 4ꢀisopropylcinnamic acid (Scheme 2).
Oxidation of cinnamic acid derivatives by a whole-cell oxidation system
Previously, preparative product formation from 4ꢀhydroxycinnamic and 3ꢀhydroxycinnamic acids
has been reported using wholeꢀcell oxidation systems consisting of CYP199A2 and a combination
23
of the electron transfer partners, palustrisredoxin, Pux, and putidaredoxin reductase. Small scale
−
1
(
250 ꢀL), high cell density (50 g cell wet weight L ) cultures were reported to generate 1 mM 3,4ꢀ
dihydroxycinnamic acid from 4ꢀhydroxycinnamic acid in 3 hours with the wildꢀtype CYP199A2
−
1
enzyme. Larger scale turnovers (50 mL, 50 g cell wet weight L ) with the same system and
substrate were reported to generate 3 mM product in 24 hours. In order to investigate the wholeꢀcell
oxidation of cinnamic acid substrates, we utilised a system developed in our laboratory which coꢀ
4
expresses CYP199A4, HaPux and HaPuR in E. coli. Using larger volume (35 mL), low cell
−
1
density cultures (3 g cell wet weight L ) and adding substrate (concentration 2 mM) with an
additional equal aliquot being added at 4 hours, we found that all the substrates yielded the same
products found in the in vitro turnovers (Scheme 1, Fig. 7 and Fig. S7).
In the CYP199A4 wholeꢀcell turnovers of 4ꢀmethylcinnamic acid and 4ꢀmethoxycinnamic
acid, all of the substrate (>95%) was converted to product at both 4 and 24 hours (Fig. 7 and S7). 3ꢀ
Hydroxyꢀ4ꢀmethoxycinnamic and 3ꢀ(4′ꢀmethoxyphenyl)propionic acids were the only other
substrates turnovers where all of the material added (2 mM) was consumed within 4 hours (Fig. 7
and S7). However, only in the turnover of 3ꢀhydroxyꢀ4ꢀmethoxycinnamic acid was all the
additional substrate consumed after 24 hours. Significant levels of the substrate (~20%) remained in
the 3ꢀ(4ꢀmethoxyphenyl)propionic acid turnover after 24 hours (Fig. S7). In the turnover of 2,4ꢀ
1
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dimethoxycinnamic acid, most of the substrate (~75%) was depleted and the product formation was
high after 4 hours but a higher ratio of substrate to product was observed after 24 hours (Fig. S7).
The turnover of 3,4ꢀdimethoxycinnamic acid resulted in conversion of approximately half the
substrate in the first 4 hours (∼960 ꢀM 3ꢀmethoxyꢀ4ꢀhydroxycinnamic acid), and a similar
proportion of starting material and product was observed at 24 hours (Fig. S7). Oxidation of 4ꢀ
methoxyphenylacetic acid by CYP199A4 generated the next highest level of product with 320 and
5
10 ꢀM of 4ꢀhydroxyphenylacetic acid being obtained after 4 and 24 hours, respectively (Fig. S7).
The levels of product in the turnovers in which the substrate lacks an alkyl or methoxy paraꢀ
substituent, were very low (≤ 120 ꢀM after 24 hours). No monooxygenase product was observed at
either 4 or 24 hours with cinnamic, 3ꢀhydroxycinnamic and 4ꢀhydroxyꢀ3,5ꢀdimethoxycinnamic
acids (Fig. S7). There was no demethylated metabolite in the turnover of 3,5ꢀdimethoxycinnamic
acid after 4 hours but low levels of product (65 ꢀM) were observed after 24 hours (Fig. S7). Low
levels of 3,4ꢀdihydroxycinnamic acid were found in the wholeꢀcell oxidation of 4ꢀhydroxycinnamic
acid at both 4 and 24 hours (∼30 and 120 ꢀM, respectively, Fig. S7). Control experiments showed
that no product was formed in the wholeꢀcell system when the P450 was excluded (Fig. S8).
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Discussion
The tight binding of 4ꢀmethoxycinnamic acid to CYP199A4 was somewhat surprising as while 4ꢀ
methoxybenzoic acid binds well to this enzyme small changes at the carboxylate group have been
shown to result in a large reduction in binding affinity with even 4ꢀmethoxyphenylacetic acid being
a poor substrate. The planar nature of 4ꢀmethoxycinnamic acid seems to be important for substrate
binding. As with 4ꢀmethoxybenzoic, 2ꢀnaphthoic and indoleꢀ6ꢀcarboxylic acids the planarity of the
substrate may maintain the interactions of the carboxylate group with the arginine and serine
5
, 6, 16, 18
residues as well as the hydrophobic contacts (Fig. 1).
Overall, the results with the cinnamic
acids reinforce the importance of the presence and the identity of the functional group at the paraꢀ
position. The trends in the activity and product selectivity of CYP199A4 broadly agree with those
obtained with similarly substituted methoxybenzoic acid substrates with low amounts of product
being formed in the absence of a 4ꢀmethoxy substituent. For example, methoxy and alkyl groups
are preferred over a hydroxyl moiety or no substitution at all.
The turnover of 4ꢀisopropylcinnamic acid generated products arising from hydroxylation at
both the αꢀ and βꢀcarbons. Metabolites arising from desaturation pathways were also observed, in
agreement with the products reported from the oxidation of 4ꢀisopropylbenzoic acid by CYP199A4.
Where the cinnamic acid substrate lacked a substituent or contained a hydroxy group at the paraꢀ
position the turnovers generated very low levels of product highlighting the selectivity of the
enzyme for this position. It may be the case that substrates containing a paraꢀhydroxy substitution
are close enough coordinate to the hemeꢀiron which may interfere with the observation of a type I
spin state shift and the commencement of the P450 catalytic cycle. In the turnover of 3,5ꢀ
dimethoxybenzoic acid by CYP199A4, a small amount of 3ꢀhydroxyꢀ5ꢀmethoxybenzoic acid was
observed. It has been hypothesised that this arises from a substrate methoxy group being oriented
20
toward the hemeꢀiron and therefore close enough to be oxidised. If only one substituent is present
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at the 3ꢀposition, it is likely to point away from the heme in a similar orientation to that observed in
5
the crystal structure of 3,4ꢀdimethoxybenzoic acidꢀbound CYP199A4 (PDB code; 4EGN).
The impact of additional substitutions in the cinnamic acid structure on substrate binding to
and the activity of CYP199A4 is generally representative of what was previously observed with
4
, 5, 20, 22
substituted benzoic acids.
However, the punitive effect of the additional substitutions at the
3
ꢀposition of the cinnamic acids was greater than the equivalent modification of benzoic acids and
this may be related to the larger size of the cinnamic acids. The larger trimethoxycinnamic acids
seem to be too bulky for the substrate binding pocket of CYP199A4 or their shape many force the
substrate to bind in such a fashion that the water bound to the heme iron is not displaced.
Overall, the trend in the levels of product formed in the in vitro CYP199A4 turnovers were
mirrored in the wholeꢀcell assays. Complete conversion of suitable substrates at concentrations up
to 4 mM could be achieved using low cell density wholeꢀcell oxidation in shake flasks. There was
higher than expected levels of product observed in the wholeꢀcell turnovers of 4ꢀhydroxycinnamic
acid when compared to the in vitro turnover studies. Recent work has shown that the E. coli
hydroxylase complex, HpaBC, is capable of oxidising this substrate to 3,4ꢀdihydroxycinnamic acid
2
9
which may account for some of this. However control wholeꢀcell experiments in the absence of
the P450 showed that no caffeic acid was being produced (Fig. S8).
The selectivity of CYP199A4 oxidation of the cinnamic acids for the paraꢀsubstituent is
interesting as chemical oxidants might be expected to target the more reactive alkene group. Indeed
3
0
chloroperoxidase oxidises cinnamic acid and its derivatives at the alkene group. Cinnamic acids
and their derivatives are phenylpropanoids which are naturally found in plants. They are constituent
components of the biosynthetic pathways of many natural products encompassing coumarins and
2
5, 26
flavonoids.
They have a range of medicinal properties including anticancer, antimicrobial and
2
5
antioxidant activities. Therefore new biocatalytic methods to facilitate the synthesis of cinnamic
derivatives could enhance their use and effectiveness. For example Ozagrel, an antiplatelet agent
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31
inhibiting thromboxane A2, is related to 4ꢀmethylcinnamic acid. CYP199A4 could have a role as
a biocatalyst for the modification of substituted cinnamic acid derivatives and the oxidation of these
substrates could be improved using protein engineering techniques to optimise the activity. For
example the F185L mutant of CYP199A2 is reported to give increased levels of product with 4ꢀ
23
hydroxycinnamic acid and is capable of oxidising cinnamic acid to 3,4ꢀdihydroxycinnamic acid.
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Experimental
General reagents, organic substrates and N,Oꢀbis(trimethylsilyl)trifluoroacetamide with
trimethylsilyl chloride (BSTFA + TMSCl, 99:1) were from SigmaꢀAldrich, TCI, Fluorochem or
VWR. Buffer components, NADH and isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG) were from
Astral Scientific, Australia. UV/Vis spectra and spectroscopic activity assays were recorded at 30 ±
0
.5 °C on an Agilent CARYꢀ60 or Varian CARYꢀ5000 spectrophotometer. Analytical High
Performance Liquid Chromatography (HPLC) was performed on an Agilent 1260 Infinity Pump
equipped with an autoinjector connected using an Agilent Eclipse Plus C18 column (250 mm x 4.6
mm, 5 ꢀm) or a Jupiter C18 300A column (250 x 4.6 mm, 5 ꢀm). The products were separated
using a gradient between 20ꢀ50% acetonitrile in water (0.1% trifluoroacetic acid (TFA) at a flow
−
1
rate of 1 mL min over 30 minutes. Gas ChromatographyꢀMass Spectrometry (GCꢀMS) data were
collected on a Shimadzu GCꢀ17A using a QP5050A GCꢀMS detector and a DBꢀ5 MS fused silica
column (30 m x 0.25 mm, 0.25 ꢀm). The injector and interface were maintained at a constant
temperature of 250 °C and 280 °C. The oven temperature was held at 100 ºC for 1 min and then
−1
increased at 15 ºC min up to 220 ºC. The HPLC retention times for the substrates and products
and the GC retention times of their trimethylsilyl (TMS) derivatives are given in the supporting
information (Table S1 and S2).
The expression and purification of CYP199A4, HaPux and HaPuR have been described
4
ꢀ6
−1
−1
elsewhere. The CYP199A4 protein concentration was calculated using ε419 = 119 mM cm as
5
previously described.
Substrate binding: spin state determination and binding titrations
The highꢀspin heme content was determined using an enzyme concentration of 1.8 – 3.7 µM in 50
mM Tris, pH 7.4, with the addition of small aliquots of substrate (from a 100 mM stock) until the
spectrum did not change. The percentage shift was estimated (to approximately ±5%) by
comparison with a set of spectra generated from the sum of the appropriate percentages of the
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spectra of the substrateꢀfree (>95% lowꢀspin, Soret maximum at 418 nm) and camphorꢀbound
(
>95% highꢀspin, Soret maximum at 392 nm) forms of WT CYP101A1.
For dissociation constant determination CYP199A4 was diluted to 1.3 – 5.5 µM using 50
mM Tris, pH 7.4, in 2.5 mL and 0.5 – 2 µL aliquots of the substrate were added using a Hamilton
syringe from 1, 10 or 100 mM stock solutions in ethanol or DMSO (Fig. 3 and S2). The maximum
Soret band peakꢀtoꢀtrough difference (ꢁA) in absorbance was recorded between 700 nm and 250
nm. Further aliquots of substrate were added until the peakꢀtoꢀtrough difference of the Soret band
d
did not change. The dissociation constants, K , were obtained by fitting ꢁA against total substrate
concentration [S] to a hyperbolic function:
ꢁ
Amax ×[S]
ꢁ
A =
K +[S]
d
where ꢁAmax is the maximum absorbance difference. 4ꢀMethoxyꢀ and 4ꢀisopropylꢀcinnamic acids
exhibited tight binding, with K < 5 µM and their data were fitted to the tight binding quadratic
d
3
2
equation:
2
(
[E]+[S]+ K ) − {([E]+[S]+ K ) − 4[E][S]}
ꢁ
Amax
A
d d
=
ꢁ
2[E]
where ꢁAmax is the maximum absorbance difference and [E] is the enzyme concentration.
Activity assays
In vitro NADH turnover rate assays were performed with mixtures (1.2 mL) containing 50 mM Tris,
pH 7.4, 0.5 ꢂM CYP199A4, 5 ꢂM HaPux and 0.5 ꢂM HaPuR. The buffer solution was saturated
with oxygen just before addition of the enzymes and the mixtures equilibrated at 30 °C for 2 min.
Substrates were added as a 100 mM stock solution in ethanol or DMSO to a final concentration of 1
mM (1% of the total volume). NADH was added to ca. 320 ꢂM (final A340 = 2.00) and the
absorbance at 340 nm was monitored. The rate of NADH oxidation was calculated using ε340 = 6.22
–1
–1
mM cm . For 3,4ꢀ(methylenedioxy)cinnamic acid which absorbed strongly at 340 nm, the reaction
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was monitored using the NADH absorption at 370 nm, where the substrate and product absorbance
–1
–1
did not interfere, using ε370 = 2.59 mM cm . Product formation rates and coupling efficiencies
were determined by analysing the amount of product formed during the turnover. Calibrations of
fixed amounts of product standards were used to quantitate the level of product by HPLC or GCꢀMS
(
vide infra).
Analysis of metabolites
The majority of the products from the enzyme turnovers were identified via coꢀelution of authentic
standards (Scheme 1) using HPLC analysis or via GCꢀMS of the trimethylsilyl chloride (TMSCl)
4
derivatised forms. After the NADH had been consumed in substrate oxidation incubations, 150 ꢂL
of the reaction mixture was mixed with 2 ꢂL of an internal standard solution (20 mM 9ꢀ
hydroxyfluorene in ethanol). This was mixed with 50 ꢂL of acetonitrile before analysis by HPLC.
The HPLC retention times for the substrates and products are given in the supporting information
(
Table S1).
For gas chromatography analysis, 990 ꢂL of the reaction mixture was mixed with 10 ꢂL of
an internal standard solution (20 mM 9ꢀhydroxyfluorene) and 3 ꢂL of 3 M HCl. The mixture was
extracted three times with 400 ꢂL of ethyl acetate and the organic extracts were combined and dried
4
over MgSO . Solvent was evaporated under a stream of nitrogen and the sample dissolved in 150 ꢂL
acetonitrile. Excess (35 ꢂL) BSTFA + TMCS (99:1) was added and the mixture left for at least 120
min to produce the trimethylsilyl ester of the carboxylic acid group and trimethylsilyl ether of the
alcohol, if formed. These reaction mixtures were used directly for GCꢀMS analysis. The retention
times for the trimethylsilyl (TMS) derivatives are given in the supporting information (Table S2).
In order to isolate and identify products for which no standards were available, and to assess
the activity of the CYP199A4 system in E. coli, we utilised a wholeꢀcell oxidation system
comprising of the plasmids pETDuetHaPux/HaPuR and pRSFDuetHaPux/CYP199A4, the
4
construction and use of has been described previously. Protein production was induced at late log
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phase by adding IPTG (to 0.1 mM final concentration) and cooling the growth to room temperature.
−
1
The cells from a 250 ml growth (~ 6 g cell wet weight L ) were harvested via centrifugation (5,000
g, 10 min) and used immediately. They were resuspended in double the volume of E. coli minimal
2 4 2 4 3 4 2 4 4
media (EMM; K HPO 7 g, KH PO 3 g, Na citrate 0.5 g, (NH ) SO 1 g, MgSO 0.1 g, 20 %
33
glucose (20 mL) and glycerol (1 % v/v) in one litre). For small scale turnovers this cell suspension
was split into 30 mL aliquots in 250 mL Erlenmeyer flasks. The substrates were added (from a 100
mM stock) to the resuspended cells to a concentration of 2 mM and the reactions were then shaken
at 30 °C and 200 rpm. Samples for HPLC analysis were taken after 4 hours at which point a further
2
mM aliquot of substrate was added and a second sample was taken after 24 hours. The
supernatant was separated from the cells by centrifugation before analysis. Control experiments in
the absence of the plasmid containing the CYP199A4 gene were performed with cell containing just
the pETDuetHaPux/HaPuR using the same method as above.
For large scale growths 200 mL of the cell suspension was added to a 2 L baffled flask and
2
mM aliquots of substrate were added at 1, 3 and 6 hours. After 20 hours, the supernatant (200
mL) was acidified, extracted in ethyl acetate (3 x 100 mL), washed with brine (100 mL) and dried
with MgSO . The organic extracts were pooled and the solvent was removed by vacuum distillation
4
and then under a stream of nitrogen. The products were purified using an Agilent 1100 HPLC
equipped with Supelcosil LCꢀ18 semiꢀprep column (5 ꢂm particle size, 25 cm × 10 mm) and a
fraction collector. A gradient, 20 ꢀ 50% of acetonitrile (with trifluoroacetic acid, 0.1%) in water
(
TFA, 0.1%) was used with UV detection at 240, 254 and 280 nm. Those fractions containing a
single product (≥95%) were combined for characterisation. The solvent was removed under reduced
pressure and the purified product was dissolved in deuterated DMSO. NMR spectra were acquired
1
13
on an Agilent DD2 spectrometer operating at 500 MHz for H and 126 MHz for C. A combination
1
13
of H and C experiments were used to determine the structures of the products (see supporting
information).
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Conclusion
The CYP199A4 product formation activity varied considerably across the range of cinnamic acid
derivatives. 4ꢀMethoxycinnamic acid was a better substrate than 3ꢀ(4ꢀmethoxyphenyl)propionic
acid which was superior to 4ꢀmethoxyphenylacetic acid. Suitably paraꢀsubstituted cinnamic acids
were excellent substrates and their turnover was selective for oxidation at the paraꢀposition. These
substrates were also found to bind with high affinity. Wholeꢀcell oxidation systems were used to
generate significant levels of product for 3ꢀ(4ꢀmethoxyphenyl)propionic acid and similarly
substituted cinnamic acids. The regioselectivity was high with the substituent at the 4ꢀposition
being the sole site of oxidation when a suitable functional group was present. Cinnamic acids
without an alkyl or alkyloxy para substituent were poor substrates generating no detectable product.
The exception being 3,5ꢀdimethoxycinnamic acid was oxidised inefficiently and slowly. This
selectivity could be useful in synthetic applications, such as selective hydroxylation and/or
deprotection reactions. As a result the biocatalytic scope of CYP199A4 has been expanded and this
knowledge can be used to improve substrate binding and turnover of this enzyme for nonꢀnatural
substrates. Furthermore in the future the enzyme could be engineered via rational mutagenesis or
directed evolution to enhance its activity and broaden its substrate range.
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Acknowledgment
This work was supported by ARC grant DP140103229 (to JJDV and SGB). SGB acknowledges the
ARC for a Future Fellowship (FT140100355). The authors also thank the University of Adelaide
for the award of a M. Phil Scholarship to RRC.
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References
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P. R. Ortiz de Montellano, ed., Cytochrome P450: Structure, Mechanism, and Biochemistry
Kluwer Academic/Plenum Press, New York, 2005.
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3
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P. R. Ortiz de Montellano, Chem. Rev., 2010, 110, 932ꢀ948.
A. Sigel, H. Sigel and R. Sigel, eds., The Ubiquitous Roles of Cytochrome P450 Proteins, John
Wiley & Sons, Weinheim, 2007.
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S. G. Bell, A. B. Tan, E. O. Johnson and L. L. Wong, Mol. Biosyst., 2010, 6, 206ꢀ214.
S. G. Bell, W. Yang, A. B. Tan, R. Zhou, E. O. Johnson, A. Zhang, W. Zhou, Z. Rao and L. L.
Wong, Dalton Trans., 2012, 41, 8703ꢀ8714.
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S. G. Bell, R. Zhou, W. Yang, A. B. Tan, A. S. Gentleman, L. L. Wong and W. Zhou,
Chemistry, 2012, 18, 16677ꢀ16688.
S. G. Bell, J. H. McMillan, J. A. Yorke, E. Kavanagh, E. O. Johnson and L. L. Wong, Chem.
Commun., 2012, 48, 11692ꢀ11694.
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61ꢀ574.
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F. P. Guengerich, Chem. Res. Toxicol., 2001, 14, 611ꢀ650.
S. G. Bell, N. Hoskins, C. J. C. Whitehouse and L. L. Wong, in Metal Ions in Life Sciences,
eds. A. Sigel, H. Sigel and R. Sigel, John Wiley & Sons, 1st edn., 2007, vol. 3, Chapter 14,
pages 437ꢀ476, p. 652.
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V. B. Urlacher and M. Girhard, Trends Biotechnol., 2012, 30, 26ꢀ36.
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M. T. Lundemo and J. M. Woodley, Appl. Microbiol. Biotechnol., 2015, 99, 2465ꢀ2483.
T. Furuya and K. Kino, Biosci. Biotechnol. Biochem., 2009, 73, 2796ꢀ2799.
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2
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Tables
Table 1 Substrate binding parameters and catalytic turnover activity data for CYP199A4 with various
substrates. The data are given as mean ± S.D. with n ≥ 3. The reaction mixtures (in 50 mM Tris, pH
−
1
7
.4) contained 0.5 ꢂM P450, 5 ꢂM HaPux and 0.5 ꢂM HaPuR. Rates are given as nmol nmolꢀCYP
−
1
−1
−1
min . The average background NADH consumption rate was 9.0 nmol nmolꢀCYP min . The data
21
for 4ꢀmethoxyphenylacetic acid were reported previously and are included for comparison.
a
b
Substrate
ꢀmethoxycinnamic acid
cinnamic acid
% HS
70%
<5%
20%
70%
<5%
K
d
/µM
NADH
PFR
180 ± 9
C %
32 ± 1
4
3.6 ± 0.1 560 ± 10
c
e
e
ꢀ
24 ± 1
98 ± 5
ꢀ
ꢀ
3
4
3
ꢀ(4′ꢀmethoxyphenyl)propionic acid
31 ± 1
21 ± 0.4
43 ± 3
85 ± 1
43 ± 2
42 ± 1
ꢀmethylcinnamic acid
ꢀhydroxycinnamic acid
202 ± 7
c
e
e
ꢀ
9 ± 0.1
ꢀ
ꢀ
21
4ꢀmethoxyphenylacetic acid
5%
690 ± 70
21 ± 1.5
5.0 ± 0.6
23 ± 2
c
4
3
4
3
3
3
3
3
2
2
3
ꢀhydroxycinnamic acid
0%
ꢀ
17 ± 0.5
51 ± 0.4
190 ± 13
50 ± 2
0.6 ± 0.04
6.6 ± 0.2
3.4 ± 0.2
13 ± 0.5
69 ± 3
f
,4ꢀ(methylenedioxy)cinnamic acid
ꢀisopropylcinnamic acid
30%
120 ± 2
>95% 3.4 ± 0.1
131 ± 10
c
g
g
,5ꢀdimethoxyꢀ4ꢀhydroxycinnamic acid <5%
ꢀ
ꢀ
ꢀ
c
,5ꢀdimethoxycinnamic acid
ꢀmethoxyꢀ4ꢀhydroxycinnamic acid
ꢀhydroxyꢀ4ꢀmethoxycinnamic acid
,4ꢀdimethoxycinnamic acid
,4ꢀdimethoxycinnamic acid
,3,4ꢀtrimethoxycinnamic acid
,4,5ꢀtrimethoxycinnamic acid
<5%
<5%
25%
20%
10%
<5%
<5%
ꢀ
22 ± 1
0.7 ± 0.02
3.3 ± 0.2
c
d
g
g
ꢀ
ꢀ
ꢀ
ꢀ
224 ± 13
840 ± 30
86 ± 4.5
408 ± 25
400 ± 20
441 ± 30
39 ± 2
239 ± 5
302 ± 17
282 ± 25
24 ± 1
59 ± 3
75 ± 4
64 ± 2
61 ± 3
c
ꢀ
c
d
g
g
ꢀ
ꢀ
ꢀ
ꢀ
[
a] PFR: product formation rate. [b] C: the coupling efficiency; the percentage of NADH consumed in
the reaction that led to the formation of products. [c] Not determined due to absence of spin state shift.
d] No significant NADH turnover activity was observed. [e] Minimal levels of product formation
[
were observed (Fig. S3) [f] Determined at A370 due to interfering absorption of the substrate. [g] no
significant product observed.
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Figures and Schemes
Scheme 1 The products formed from CYP199A4 enzyme turnovers with different cinnamic acid
derivatives (abbreviations; CA, cinnamic acid; MCA, methoxycinnamic acid).
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4

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Scheme 2 The products formed from CYP199A4 enzyme turnovers with 4ꢀisopropylcinnamic acid
The products were identified as; A, 4ꢀ(1′ꢀhydroxyisopropyl)cinnamic acid, B, 4ꢀ(2′ꢀ
hydroxyisopropyl)cinnamic
acid,
C,
4ꢀ(propꢀ1′ꢀenꢀ2′ꢀyl)cinnamic
and
D,
4ꢀ(1′,2′ꢀ
epoxyisopropyl)cinnamic acid.
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Figure 1 The active site of 4ꢀmethoxybenzoic acidꢀbound CYP199A4 (PDB code 4DO1). The amino
acid residues which confer the substrateꢀspecificity of the enzyme have been shown. 4ꢀ
Methoxybenzoic acid is shown in yellow, the heme in grey and the amino acids in green.
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Figure 2 The substrates tested for binding and activity with CYP199A4.
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Figure 3 Spin state shift of CYP199A4 with the substrates (a) 3ꢀ(4′ꢀmethoxyphenyl)propionic acid, (b)
ꢀmethoxycinnamic acid, (c) 4ꢀmethylcinnamic acid and (d) 4ꢀisopropylcinnamic acid. Red; substrateꢀ
bound CYP199A4, black; substrateꢀfree CYP199A4.
a) (b)
4
(
(c)
(d)
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DOI: 10.1039/C6RA11025H
Figure 4 Dissociation constant analyses of selected substrates with CYP199A4. The protein
concentration, the absorbance of the peak and trough used to calculate ꢁA and the K are also
provided. (a) 3ꢀ(4′ꢀmethoxyphenyl)propionic acid (CYP199A4 concentration, 2.1 ꢀM, A390ꢀA421 and
31 ꢀM) (b) 4ꢀmethoxycinnamic acid (CYP199A4 concentration, 1.3 ꢀM, A390ꢀA421 and K 3.6 ꢀM)
and (c) 4ꢀmethylcinnamic acid (CYP199A4 concentration, 1.5 ꢀM, A390ꢀA421 and K 21 ꢀM).
a) (b)
d
K
d
d
d
(
(c)
2
9