Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1

Article abstract of DOI:10.1002/1873-3468.12519

Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP260B1 by site-directed mutagenesis to disrupt the substrate positioning and proton delivery, respectively. Furthermore, we used the kinetic solvent isotope effect on the steady-state turnover of the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.

Full text of DOI:10.1002/1873-3468.12519

Received Date : 27-Oct-2016
Revised Date : 25-Nov-2016
Accepted Date : 01-Dec-2016
Article type : Research Letter
Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by
myxobacterial CYP260B1
Martin Litzenburger, Roberta Lo Izzo, Rita Bernhardt, and Yogan Khatri
Institut für Biochemie, Universität des Saarlandes, Campus B.2.2, 66123, Saarbruecken, Germany
Corresponding author:
Yogan Khatri, Saarland University, FR 8.3
Biochemistry, Building B2.2, 66123 Saarbruecken
E-mail: yogan.khatri@uni-saarland.de
Tel: 0049 681 302 7762
Fax: 0049 681 302 4739
Abbreviations:
AdR, Adrenodoxin reductase; Adx4-108, Adrenodoxin (truncated form); KSIE, Kinetic Solvent Isotope
Effect; P450, cytochrome P450.
Keywords:
Aldehyde oxidation, CYP260B1, Kinetic Solvent Isotope Effect, Sorangium cellulosum So ce56
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lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/1873-3468.12519
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Abstract:
Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde
dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we
demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP260B1.
Following our docking studies of the aldehyde, we generated T224A and T234A mutants of
CYP260B1 by site-directed mutagenesis to disrupt the substrate positioning and proton delivery,
respectively. Furthermore, we used the kinetic solvent isotope effect on the steady state turnover of
the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results
suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.
Introduction:
Cytochromes P450 are heme-thiolate containing monooxygenase enzymes found in all domains of life
[
1]. They catalyze a broad range of different reactions such as hydroxylation, epoxidation,
dealkylation or C-C cleavage [2-4]. P450s are also able to oxidize aldehydes to the corresponding
acids [5].
There are several studies described for the oxidation of saturated, α,β- unsaturated or α,β- branched
aldehydes by P450s [5-10]. In addition, some studies also describe the decarbonylation and the
formation of olefins for branched compounds such as cyclohexanecarboxaldehyde or methylated
propionaldehydes [11,12]. The utilization of α,β-unsaturated aldehydes as substrates resulting
predominantly into carboxylic acids was also shown for several P450s [5]. Interestingly, most of these
studies were performed with xenobiotic metabolizing P450s such as CYP2B4, CYP1A2 or CYP2E9
[
5,12]. In contrast, only little is known concerning aldehyde oxidation by microbial P450s. The most
prominent example for a bacterial P450 capable of aldehyde oxidation is CYP102A1 (P450BM3) and
its oxidation of ω-oxo fatty acids to the corresponding dicarboxylic acids [8], whereby the reactive
species has not been studied.
Two different reaction mechanisms are proposed to catalyze such an oxidation reaction, either by the
hydrogen abstraction and rebound hydroxylation mechanism or by nucleophilic attack of the ferric
peroxoanion [13] (see Scheme 1).
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Scheme 1: Proposed mechanisms for the oxidation of aldehydes to the corresponding acids. The nucleophilic attack
by ferric peroxoanion (A), and the hydrogen abstraction and rebound reaction (B) are shown.
As shown in Scheme 1, pathway A is independent of protons whereas pathway B is requiring protons
for the product formation. To distinguish between these two pathways, the kinetic solvent isotope
effect can be used [14,15]. Furthermore, the proton delivery inside the active site can also be disrupted
by site-directed mutagenesis and replacing the corresponding amino acids [16-18]. This approach was
previously performed for mammalian CYP2B4 by creating the T302A variant to identify the reactive
species capable of the conversion of aldehydes [6].
In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by myxobacterial
CYP260B1. In addition, docking of cinnamaldehyde into the crystal structure of CYP260B1 was
performed followed by the creation of threonine mutants (T224A, T232A and T224A/T232A). We
investigated the KSIE for the oxidation reaction not only for the wild type but also for the created
threonine mutants T224A and T232A that are proposed to be involved in the substrate orientation and
proton delivery, respectively.
Material and Methods:
Chemicals:
Isopropyl β-D-1-thiogalactopyranoside and 5-aminolevulinic acid were purchased from Carbolution
Chemicals (Saarbruecken, Germany). Bacterial media were obtained from Becton Dickinson
(
Heidelberg, Germany). All other chemicals were purchased from standard sources in the highest
purity available.
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Strains:
The E. coli strain Top 10 for cloning purpose was obtained from Invitrogen (San Diego, USA). The E.
coli strain C43(DE3) for the heterologous expression of the P450s was purchased from Novagen
(
Darmstadt, Germany).
Docking studies:
The automated docking program AUTODOCK (version 4.20) [19,20] was applied for docking of
cinnamaldehyde into the substrate free crystal structure of CYP260B1 (PDB 5HIW) [21]. The
Windows version 1.5.6 of Autodock Tools was used to compute Kollman charges for the enzyme
CYP260B1 and Gasteiger-Marsili charges for the ligands [22]. A partial charge of +0.400e was
assigned manually to the heme-iron, which corresponds to Fe(II) that was compensated by adjusting
the partial charges of the ligating nitrogen atoms to −0.348e. Flexible bonds of the ligands were
assigned automatically and verified by manual inspection. A cubic grid box (56×56×56 points with a
grid spacing of 0.375 Å) was centered 5 Å above the heme-iron. 100 docking runs were carried out
applying the Lamarckian genetic algorithm using default parameter settings.
Site-directed mutagenesis of CYP260B1:
Targeted exchange of single amino acids was undertaken by QuikChange® mutagenesis with Pfu
polymerase following manual instructions form Agilent Technologies (Santa Clara, USA). The
corresponding forward and reversed primers for the T224A, T232A variants and the T224A/T232A
double mutant are shown in supplemental Table 1. The sequences of the mutants were verified by
automated sequencing (Eurofins Genomics, Ebersberg, Germany).
Expression and purification of the enzymes:
The expression and purification of CYP260B1 and its mutated variants was performed as described
previously [21]. The truncated bovine adrenodoxin (Adx4-108) and adrenodoxin reductase were
expressed and purified as described elsewhere [23,24].
Spectrophotometric characterization:
UV–visible spectra for the purified P450s were recorded at room temperature with a double-beam
spectrophotometer (UV-2101PC; Shimadzu, Kyoto, Japan). The proteins were diluted in 50 mM Tris-
HCl buffer (pH 7.4) with 2% glycerol and the concentration of the P450s was estimated by CO
-1
-1
difference spectroscopy assuming Δε (450–490) = 91 mM * cm according to the method of Omura
and Sato [25]. Additionally, the P450s were measured in the reduced form as well as in the substrate
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bound form. The proteins were reduced by the addition of a few grains of sodium dithionite and
analyzed in the range of 300-700 nm. For the measurement of the substrate bound form, the
corresponding substrates were added in excess (2-5 times) and also analyzed in the range of 300-700
nm.
In vitro conversions and steady state kinetic turnover:
A reconstituted in vitro system containing the corresponding CYP260B1 variant (0.3 µM), AdR (0.9
µM) and Adx4-108 (6 µM) in a final volume of 500 µl of Tris buffer (50 mM, pH 7.4) with 2% glycerol
was used. Cinnamaldehyde (dissolved in EtOH) was added to a final concentration of 100 µM. The
reaction was started by the addition of NADPH (250 µM). After 30 min at 30°C the reaction was
quenched by adding 10 µl HCl (1:1 diluted with water). The aqueous phase was extracted twice with
chloroform (2 x 500 µl). A negative control in the absence of P450 in the reaction sample was
employed for each substrate to verify the P450-dependent reaction. Likewise, the conversion of
progesterone (200 µM final concentration) was performed except that samples were extracted
immediately without acidification.
The steady state kinetic turnover measurements of cinnamaldehyde were performed in protiated and
deuterated solvent systems. To investigate the effect of radical scavengers on the catalytic rate, the in
vitro conversions were performed with the addition of ascorbate (20 mM), catalase (20 U) and
superoxide-dismutase (3 U), individually as well as in combination.
HPLC analyses:
HPLC analyses were performed on a system consisting of a PU-2080 HPLC pump, an AS-2059-SF
autosampler, and a MD-2010 multi wavelength detector (Jasco, Gross-Umstadt, Germany). A
Nucleodur 100–5 C18 column (125 x 4 mm, Macherey–Nagel, Düren, Germany) was used at 40°C.
Water with 0.1% trifluoroacetic acid (A) and acetonitrile with 0.1% trifluoroacetic acid (B) were used
as mobile phases. A gradient from 25% to 55% B over 10 min was used for separation. For
quantification of the compounds calibration curves in the range of 0 to 200 µM were used. HPLC
analyses of progesterone were performed with 10% acetonitrile in water (A) and acetonitrile (B) as
mobile phases. A gradient of 10 to 60% B over 30 min was used for separation of the analytes.
Whole-cell conversions:
The whole-cell conversion was performed according to the method previously described [26].
Cinnamaldehyde (200 µM) conversion was performed in 8 sealed baffled flasks (1 l) each filled with
250 ml of M9CA medium. After 48 h, the reaction was quenched and extracted two times with the
same volume of ethyl acetate. The crude extract was evaporated to dryness and stored at 4°C until
purification.
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Purification of the product:
The extract of the cinnamaldehyde conversion was purified by silica gel chromatography (hexane:
ethyl acetate – 7:3). Fractions were collected and analyzed by thin layer chromatography. An UV
lamp at 254 nm was used for the detection of the desired product. Product containing fractions were
pooled and the solvent was removed by vacuum evaporation. About 23 mg of a light yellowish solid
were obtained.
NMR analysis:
NMR spectra were recorded on a Bruker (Rheinstetten, Germany) DRX 500 NMR spectrometer. A
1
13
combination of H, C, and HSQC experiments was used for structure elucidation. All chemical shifts
1
13
are relative to CHCl (δ=7.24 for H NMR) or CDCl (δ=77.00 for C NMR) using the standard δ
3
3
notion in parts per million (ppm).
1
trans-cinnamic acid: H NMR (CDCl , 500 MHz): 7.62 (d, 1H, J=16.0 Hz, vinylic C-H), 7.38 (m, 2H,
3
1
3
aromatic C-H), 7.23 (m, 3H, aromatic C-H), 6.29 (d, 1H, J=16.0 Hz, vinylic C-H); C NMR (CDCl ,
3
1
1
25 MHz): 172.1 (C=O), 147.0 (C-H), 134.0 (C), 130.7 (C-H), 128.9 (C-H, 2x), 128.3 (C-H, 2x),
17.3 (C-H).
Results:
Substrate identification and product elucidation:
Since CYP260B1 from S. cellulosum So ce56 is known for the conversion of small compounds such
as ionones and damascones [27], we tested cinnamaldehyde, a molecule of similar size containing an
α, β-unsaturated aldehyde group. This compound served as a substrate and the conversion showed a
single product (see Figure 1). With our previously established whole-cell system [26], we were able to
produce a sufficient amount of product for NMR analysis. The obtained product was identified as
cinnamic acid.
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Figure 1: The chromatogram shows the conversion of cinnamaldehyde (t = 8.4 min) to cinnamic acid (t = 6.9 min)
R
R
catalyzed by CYP260B1.
Site-directed mutagenesis
Very recently, we have elucidated the crystal structure of CYP260B1 [21]. In order to get a deeper
insight into the active site of this enzyme and the substrate orientation, we performed docking studies
of cinnamaldehyde into the substrate-free crystal structure of CYP260B1 (see Figure 2). The docking
study showed that threonine 224 is suggested for the positioning of cinnamaldehyde. In addition, the
amino acid sequence alignment of CYP260B1 with other P450s showed that threonine 232 is the
conserved residue found in most P450s and is located in the I-helix next to a glutamic acid. This acid-
alcohol pair is supposed to be involved in the proton delivery and catalysis [28]. The involvement of
the conserved threonine in proton delivery was previously exemplified for the mutant T252A of
CYP101A1 (P450cam) [29] or T306A of CYP17A1 [16].
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Figure 2: Docking of cinnamaldehyde (green) into the substrate free crystal structure of CYP260B1. The I-helix
highlighting T224, E231 and T232 (blue cartoon) above the heme-plane (brown) is shown.
To study the effect of T224 and T232, both amino acids were individually replaced by alanine
resulting into the T224A and T232A variants. In addition, a double mutant in which both threonines
were replaced by alanine (T224A/T232A) was also created.
Characterization of the mutants by UV-Vis spectroscopy and SDS-PAGE:
The purified protein mutants were analyzed by SDS-PAGE and showed a molecular mass of about 50
kDa (see supplemental Figure 1). Furthermore, the enzymes were characterized by UV-Vis
spectroscopy (see Supplemental Table 2 and Supplemental Figure 2).
Functional characterization of the mutants by conversions of cinnamaldehyde and progesterone:
To identify the functional properties of the novel CYP260B1 variants, conversions were performed
with cinnamaldehyde as well as the known substrate progesterone [21]. This steroid was chosen for
the functional characterization, since this compound was converted more efficiently by CYP260B1
compared with carotenoid-derived aroma compounds.
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Table 1: Conversions of cinnamaldehyde and progesterone by CYP260B1, T224A mutant, T232A mutant and
T224A/T232A double mutant.
Relative conversion by [%]
Substrate
CYP260B1
T224A mutant
T232A mutant
T224A/T232A
(
control)
100
double mutant
Cinnamaldehyde
Progesterone
52
84
9
64
12
100
100
As shown in Table 1, all variants were able to convert both substrates; however, the product yields
differed strongly. The conversion of cinnamaldehyde was decreased to almost half of the activity of
the wild type by the T224A variant. The predicted substrate orientation indicated that the phenyl ring
is placed towards the heme-plane, which is not in accordance with the formed product. Nevertheless,
this amino acid influenced the productivity towards cinnamic acid demonstrating its function in
product formation. CYP260B1 and the T224A mutant converted the same amounts of progesterone
but the product pattern was shifted towards a higher selectivity for T224A (see Supplemental Figure
3), which is in agreement with our assumption that this amino acid is important for substrate
orientation.
The conversion of cinnamaldehyde was only slightly decreased by the T232A mutant. In contrast, the
~
activity of this mutant towards the conversion of progesterone was significantly impaired ( 10%)
compared with the activity of the wild type. This observation suggests that the steroid conversion is
dependent on the presence of protons, whereas the oxidation of cinnamaldehyde seems to be
independent on the presence of protons.
The double mutant (T224A/T232A) reflected the effects of the single mutants, in which the
progesterone conversion was as strongly inhibited as shown for the T232A mutant. For the
cinnamaldehyde conversion, the double mutant showed a similar effect as the T224A mutant;
however, its activity was still higher than that of the T224A variant and might be explained by some
conformational changes inside the active site caused by replacing two threonines with the smaller
amino acid alanine. The spectrum of the double mutant in cinnamaldehyde-bound form showed a
shoulder at 393 nm (see Supplemental Table 2 and Supplemental Figure 2) representing a small type-I
shift induced by this substrate indicating a conformational change.
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Steady state turnover of cinnamaldehyde in protiated and deuterated solvent systems:
Since the conversions with the mutants indicated that cinnamaldehyde oxidation is independent on
protons, the reactions were performed in both water and deuterium oxide to determine the KSIE
(
k /k ) at steady state (see Figure 3A+B). The activity in deuterated solvent was slightly higher than
H D
the reaction in protiated solvent with an inverse KSIE of about 0.9.
Figure 3: A. Time dependent conversion of cinnamaldehyde by CYP260B1 (A). The rate of conversion of the
substrate in protiated and deuterated solvent systems (B), and the effect of radical scavengers (C) are shown. SOD,
superoxide-dismutase; RSC, radical scavengers in combination. Results are obtained from triplicate experiments ±
standard deviation.
The catalytic cycle of P450s contains three unproductive pathways, in which the electrons are
funneled for the production of superoxides (autooxidation shunt), hydrogen peroxide (peroxide shunt)
or water (oxidase shunt), respectively [28]. To investigate the potential role of a H O -mediated
2
2
conversion as well as the influence of unproductive pathways, diverse radical scavengers such as
ascorbate (neutralizing the superoxide radical, singlet oxygen and hydroxyl radicals), catalase
(
decomposing hydrogen peroxide to water and oxygen) and superoxide dismutase (scavenger of
superoxide anion) were applied individually as well as in combination. However, none of the
scavengers showed a considerable effect on the conversion rate (see Figure 3C). Additionally, the
reactions were performed in the presence of H O , since the oxidation of cinnamldehyde can be
2
2
mediated by H O in the presence of transition metals [30]. As shown in Figure 3C, H O was able to
2
2
2
2
produce low amounts of cinnamic acid, though its yield can be neglected compared to the control. All
these results suggest that the reaction is catalyzed by a nucleophilic attack of the ferric peroxoanion
and not mediated by a hydrogen abstraction and rebound mechanism.
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The effect of the KSIE was also tested for the created mutants (see Figure 4). Both the single mutants
and the double mutant were not considerably influenced by the radical scavengers (see Supplemental
Figure 4). In addition, all variants showed a higher conversion rate for the deuterated solvent system
compared with the protiated one resulting in an inverse KSIE of 0.6 and 0.9 for T224A and T232A,
respectively, and a KSIE of about 0.9 for the double mutant. The T232A variant showed a conversion
rate of ~2 nmol product per minute per nmol P450 which is similar to the conversion rate of the wild
type suggesting that a disrupted proton delivery is not influencing the activity. We observed that
neither the application of the deuterated solvent system nor employing the T232A variant or the
combination of both approaches showed a considerably decreased cinnamic acid formation. This
observation corroborated with the assumption of a nucleophilic attack by the ferric peroxoanion as
most probable reaction mechanism for the aldehyde conversion.
Figure 4: The conversion rates of T224A (A), T232A (B) and double mutant (C) in protiated and deuterated solvent
systems.
Discussion:
In plants, cinnamic acid is an important intermediate in the phenylpropanoid pathway, that is further
hydroxylated in position 4 by P450s (also known as cinnamate 4-hydroxylases) [31] resulting in p-
coumaric acid, a precoursor for the biosynthesis of several secondary metabolites such as lignins,
coumarins or flavonoids [32]. Thereby, cinnamic acid is produced by deamination of the amino acid
phenylalanine [33]. In this study, we demonstrated the formation of cinnamic acid by oxidation of
cinnamaldehyde, an important compound for the flavor and fragrance industry [34], catalyzed by
myxobacterial CYP260B1. This enzyme belongs to a novel P450 family found in Sorangium
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cellulosum So ce56 [35] and is known for the conversion of steroids and carotenoid-derived aroma
compounds [21,27]. It is not surprising that besides carotenoid-derived aroma compounds
cinnamaldehyde acts as substrate, since they are similar in size and functional groups. Interestingly,
this substrate was not functionalized in an endocyclic position as demonstrated for some carotenoid-
derived aroma compounds. Reasons for that observation might be related to the substrate orientation
caused by the structural differences such as the phenyl ring or by the presence of the highly reactive α,
β-unsaturated aldehyde moiety.
Since the oxidation of aldehydes is supposed to be catalyzed by either ferryl species or ferric
peroxoanion [13], we performed studies such as KSIE and site-directed mutagenesis to get a deeper
insight into the reactive species capable of aldehyde oxidation. The activity was thereby neither
considerably decreased by employing a deuterated solvent system nor by the T232A variant
disrupting the proton transfer. Thus, both approaches supported the assumption of a proton
independent reaction catalyzed by nucleophilic attack of the ferric peroxoanion and not by hydrogen
abstraction and rebound reaction. The functionality of the T232A variant was also tested by the
progesterone conversion, which, however, showed only about 10% of the activity compared with the
wild type. The most common reaction for steroid functionalization is the hydroxylation, which is
performed by the proton dependent ferryl species [36]. With a disrupted proton delivery to the active
site, this kind of reaction should be drastically decreased as shown here. As a result, the functionality
of this amino acid can be confirmed. Concerning the function of T224, our docking studies suggested
that this position is important for substrate positioning. This assumption was verified by the altered
product pattern of the progesterone conversion (see Supplemental Figure 3). The wild type showed a
higher selectivity towards product number 5, whereas T224A favored the formation of product 3. In
contrast, the product pattern of cinnamaldehyde conversion was not changed, although the activity
was strongly influenced proving the influence of this amino acid towards the productivity. However,
the predicted docking position cannot explain why the side chain is oxidized, since the phenyl group
seems to be centered above the heme-plane. For that reason, another substrate orientation might be
more probable for substrate oxidation or the substrate may be more mobile than indicated by the
docking study.
Several studies on various mammalian P450s have described the oxidation of structural diverse
aldehydes to their corresponding acids [5-7,9,37], among which CYP2B4 is the best studied P450,
and it was shown that the acid production is mainly catalyzed by the ferryl species by this enzyme [6].
In contrast, the peroxoanion dependent reaction led to several other products such as alcohols or
olefins. Some of the formed intermediates were also able to interact with the P450 itself leading to an
inactivation of the enzyme [6,38]. However, such an adduct based inactivation was predominantly
found at higher substrate concentrations of several hundred micromolars. During the conversion of
cinnamaldehyde by CYP260B1, neither side products nor a significant loss in its activity were
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observed, which might be related with a specific conformation of the active site hindering a
deformylation reaction. In addition, only low substrate concentrations were used for the CYP260B1-
dependent reaction.
In conclusion, these results show the oxidation of cinamaldehyde to cinnamic acid by myxobacterial
CYP260B1. Moreover, our results suggested that the ferric peroxoanion species is capable of
catalyzing this type of reaction instead of the often proposed ferryl species.
Acknowledgements:
The authors thank Birgit Heider-Lips for the purification of AdR and Adx and Dr. Josef Zapp for
measuring the NMR sample. This work is supported by a grant of the Deutsche
Forschungsgemeinschaft (Be1343/23-2) to R.B.
Author contributions:
ML, RB and YK designed the project. ML, RLI and YK carried out the experiments and analyzed the
data. ML wrote the manuscript together with contributions from RB and YK. All authors read and
approved the manuscript.
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