α-Oxidative decarboxylation of fatty acids catalysed by cytochrome P450 peroxygenases yielding shorter-alkyl-chain fatty acids

Article abstract of DOI:10.1039/c7cy02263h

Cytochrome P450 peroxygenases belonging to the CYP152 family catalyse the oxidation of fatty acids using H₂O₂. CYP152N1 isolated from Exiguobacterium sp. AT1b exclusively catalyses the α-selective hydroxylation of myristic acid at physiological H₂O₂ concentration. However, a series of shorter-alkyl-chain fatty acids such as tridecanoic acid were produced from myristic acid by increasing the concentration of H₂O₂ (1-10 mM). The yield of tridecanoic acid from myristic acid reached 17%. An ¹⁸O-labeled oxidant study suggested that CYP152N1 catalysed the overoxidation of α-hydroxymyristic acid to form α-ketomyristic acid, which in turn was spontaneously decomposed by H₂O₂ to yield tridecanoic acid. Crystal structure analysis of CYP152N1 revealed its high similarity to other CYP152 family enzymes, such as CYP152A1 and CYP152B1. MD simulations of α-hydroxymyristic acid accommodated in CYP152N1 proposed a possible pre-oxidation conformation of α-hydroxymyristic acid for the decarboxylation reaction.

Full text of DOI:10.1039/c7cy02263h

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α-Oxidative Decarboxylation of Fatty Acids Catalysed by
Cytochrome P450 Peroxygenases Yielding Shorter-Alkyl-Chain
Fatty Acids
Received 00th January 20xx,
Accepted 00th January 20xx
Hiroki Onodaa, Osami Shoji*a,b, Kazuto Suzukia, Hiroshi Sugimotob,c, Yoshitsugu Shirod, and
Yoshihito Watanabe*e.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Cytochrome P450 peroxygenases belonging to the CYP152 family catalyse the oxidation of fatty acids using H2O2.
CYP152N1 isolated from Exiguobacterium sp. AT1b exclusively catalyses the α-selective hydroxylation of myristic acid
under physiological H2O2 concentration. However, a series of shorter alkyl-chain fatty acids such as tridecanoic acid were
produced from myristic acid by increasing the concentration of H2O2 (1-10 mM). The yield of tridecanoic acid from myristic
acid reached 17%.18O-labeled oxidant study suggested that CYP152N1 catalysed the overoxidation of α-hydroxymyristic
acid to form α-ketomyristic acid, which in turn spontaneously decomposed by H2O2 to yield tridecanoic acid. Crystal
structure analysis of CYP152N1 revealed high similarity to other CYP152 family enzymes, such as CYP152A1 and CYP152B1.
MD simulations of α-hydroxy myristic acid accomodating in CYP152N1 proposed a possible pre-oxidation-conformation of
α-hydroxy myristic acid for decarboxylation reaction.
carboxylate moiety of the fatty acid interacting with Arg241
located at the distal side of the haem prosthetic group. This
arginine is highly conserved in other fatty acid peroxygenases,
Introduction
Cytochrome P450s (CYPs or P450s) are monooxygenases found
in a plethora of animals, plants and microorganisms1. P450s
catalyse the oxidation of a variety of organic compounds
related to biological, pharmaceutical, and medicinal chemistry.
Most P450s utilise molecular oxygen for monooxygenation
reactions2, in which electrons are provided by NAD(P)H to
generate the haem active oxygen species (Compound I). In
1996, Matsunaga and co-workers reported a peroxygenase
activity of Cytochrome P450SPα (CYP152B1) isolated from
Sphingomonas paucimobilis3, 4, which catalyses the α-selective
hydroxylation of long-chain fatty acids5. CYP152B1 exclusively
oxidizes fatty acids6. Moreover, the Km for H2O2 of the
peroxygenation catalysed by CYP152B1 was sufficiently low
(72 μM)7. Accordingly, CYP152B1 was classified as a fatty acid
peroxygenase (EC 1.11.2.4). The crystal structure of a fatty acid
bound-form of CYP152B1 (PDB: 3AWM)8 showed the
such as P450BSβ (
CYP152A1) isolated from Bacillus subtilis9 and
P450CLA (CYP152A2) isolated from Clostridium acetobutylicum10.
Both CYP152A1 and CYP152A2 catalyse α- and β-hydroxylation
of fatty acids11, 12. The Km of H2O2 for fatty acid hydroxylation
by CYP152A1 was estimated to be 21 μM13. Based on crystal
structure analysis of CYP152A1, a reaction mechanism for the
formation of the active oxygen species (Compound I) was
proposed, wherein the carboxylate group of the fatty acid
interacting with the distal arginine serves as an acid-base
catalyst for the activation of hydrogen peroxide (Scheme 1A)14
.
In fact, mutation of the distal arginine to alanine (R242A)
decreased both the affinity for hydrogen peroxide (Km = 4.4
mM) and the catalytic activity (kcat = 0.83 min-1)13. Recently,
Scheme 1 Compound I formation mechanisms of CYP152 family enzymes
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Quantum Mechanical/Molecular Mechanical (QM/MM) and experimental approach employing 18O-labelled hydrogen
Molecular Dynamics (MD) studies suggested another peroxide as an oxygen source in combination with MD
mechanism15 (Scheme 1B), wherein the carboxylate group of simulations based on the crystal structure of fatty acid-bound
the fatty acid plays an important role in orienting the hydroxyl form of CYP152N1.
radical (HO·) toward Compound II. The interaction of the
carboxylate moiety with the distal arginine is critical for active
Results and Discussion
oxygen species formation in both mechanisms. Previous
research on chemical rescue by acetate-anions enabling non-
UV-vis spectra and myristic acid oxidation
native substrates hydroxylation lend support to a carboxylate-
dependent mechansism16.
Fig. 1 UV-vis spectra of CYP152N1 (6.2 μM) of ferric form (solid line), ferrous form
(dotted line), CO binding ferrous form (chain line).
Scheme
2 Possible reaction mechanisms of fatty acid preoxygenation reactions
catalysed by A) CYP152A1 and B) CYP152L1
The UV-VIS spectrum of purified CYP152N1 (Fig. S1) showed a
Soret band absorption peak at 417 nm and two Q-band
absorption peaks at 542 nm and 565 nm (Fig. 1 solid line),
indicative of the typical low spin ferric state of P450s. The
absorption of dithionite-reduced ferrous-CO adducts was
observed at 444 nm (Fig. 1 chain line), indicative of thiolate
ligation to the haem iron. Upon addition of myristic acid or
palmitic acid to ferric CYP152N1, the Soret band absorption
peak slightly shifted to a shorter wavelength (Fig. S2). Type I
shifts are caused by displacement of water from the haem
resulting in a spin state change of the haem Iron from low to
high spin, indicative of positive substrate binding. Dissociation
constants (Kd) of palmitic acid and myristic acid were
estimated to be 47 μM and 6.5 μM, respectively (Fig. S3).
Although CYP152 family enzymes have been considered to
solely play a role in fatty acid hydroxylation reactions, this
paradigm came under scrutiny in 2011 with the discovery of
OleTJE (CYP152L1) isolated from Jeotgalicoccus sp. ATCC 8456,
which catalyses terminal olefin biosynthesis from fatty acids17,
18
.
The terminal olefin is produced via
20
a
fatty acid
decarboxylation reaction19,
(Scheme 2B), which can be
initiated by C-H abstraction21 in the same manner as fatty acid
hydroxylation by CYP152A1 (scheme 2A). In addition, it was
reported that CYP152A1 also produced a small amount of
terminal olefin17, while CYP152A2 does not produce any
terminal olefin22. Recently, it was reported that three CYP152
family enzymes, such as CYP-MP (CYP152P1) isolated from
Methylobacterium populi, CYP-Aa162 (CYP152A8) isolated
from Alicyclobacillus acidocaldarius, and CYP-Sm46 (CYP152L2)
isolated from Staphylococcus massiliensis, can also produce
terminal olefins23, 24. These results clearly show that CYP152
family enzymes possess potential to perform reactions other
than fatty acid hydroxylation, encouraging us to explore other
CYP152 family enzymes.
Herein we report that the α-oxidative decarboxylation
reaction of fatty acids can also be catalysed by CYP152N1
(P450Exα) isolated from Exiguobacterium sp. AT1b25, which was
obtained from the hot springs of Yellowstone National Park.
Shorter-alkyl-chain fatty acids including tridecanoic acid were
produced from myristic acid, upon increase of the hydrogen
peroxide concentration. The amino-acid sequence of P450Exα
possesses a 44% sequence identity to CYP152A1, leading to its
classification as CYP152N1. Crystal structure analysis showed
that CYP152N1 had the distal arginine which was critical for
fatty acids binding. We investigated a possible reaction
mechanism for α-oxidative decarboxylation by using an
Fig. 2 GC-MS chart (MIC m/z = 117, 147) of myristic acid oxidation in the presence of
200 μM or 5 mM H2O2. Retention time of TMS derivatives: C12 (10.0 min), C13 (11.0
min), C14 (12.0 min), C13α-OH (12.7 min), C14α-OH (13.7 min), C14β-OH (13.8 min) IS
(Internal standard, 13.9 min).
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Hydroxylation of myristic acid was examined using CYP152N1 acid was consumed by H2O2 via non-enzymatic decarboxylation
by changing the concentration of myristic acid as well as H2O2. to yield tridecanoic acid.
The GC-MS chart showed the peak of α-hydroxy myristic acid
in the presence of 200 μM hydrogen peroxide (Fig. 2A). The
appreciable peaks corresponding to β-hydroxy myristic acid or
1-tridecene was not detected. The configuration of α-hydroxy
myristic acid was confirmed by HPLC equipped with a chiral
column (Fig. S4) to be dominantly (S)-form (91%ee). The regio-
and stereo-selectivity of myristic acid hydroxylation were
similar to those of CYP152B16, 8. Michaelis-Menten kinetic
Scheme 3 Reported catalytic reaction mechanism of α-keto decarboxylation.
analysis (Fig. S5) revealed that the kcat reached 1900 ± 130 min-
1, which is higher than that of both CYP152A1 (1400 ± 180 min-
1) and CYP152B1 (1300 ± 20 min-1). The Km of CYP152N1 for
myristic acid (130 ± 20 μM) was higher than that of CYP152A1
(66 ± 20 μM) and CYP152B1 (43 ± 2 μM)8.
Interestingly, tridecanoic acid could be detected when the
concentration of H2O2 was raised to 5 mM. GC-MS analysis
allowed the detection of peaks corresponding to tridecanoic
acid, lauric acid, and α-hydroxy tridecanoic acid (Fig. 2B, S6).
The maximum yield of tridecanoic acid produced from myristic
acid in a one-minute oxidation reaction was 17% using 10 mM
Scheme 4 Proposed reaction scheme of tridecanoic acid formation.
H2O2 (Fig. 3). We also found that myristic acid was primarily
converted to decanoic acid (C10) by 20 minutes reaction using
CYP152N1 (Fig. S7).
A possible reaction scheme of tridecanoic acid formation
reactions is shown in Scheme 4. Herein, the carboxylate of
myristic acid is eliminated as carbon dioxide. To confirm the
elimination of carboxylate, the oxidation of myristic acid by
CYP152N1 was carried out using 18O-labelled hydrogen
peroxide (H218O2). Tridecanoic acid produced from myristic
acid and H218O2 contained two 18O-labelled atoms as the major
product (Fig. S9A). This result indicated C16O2 was released
from myristic acid after stepwise oxidation. Upon the oxidation
of α-hydroxy myristic acid (C14α-16OH), mono-18O-labelled
tridecanoic acid was the main product (Fig S9B). These
consistent results suggested that one oxygen atom originated
from the hydroxyl group of α-hydroxy myristic acid was
introduced to the product. The fact indicated that most of α-
keto acids were produced without going through the hydrated-
α-keto acid, which could be produced by the (R)-α-
hydroxylation of (S)-α-hydroxy myristic acid, while it was
reported that α-keto acids had chance to hydrated by water30.
A small amount of mono-18O-labelled tridecanoic acid was
Fig. 3 Fatty acids concentration of the product mixture after oxidation of myristic acid
catalysed by CYP152N1
The yield of tridecanoic acid was enhanced by increasing the produced in the presence of 18O-labelled water (Figure S10),
concentration of hydrogen peroxide accompanied by
although there was no 18O-labelled myristic acid present.
a
decrease in α-hydroxy myristic acid (Fig. 3). These results These results indicate that the α-keto acid intermediate can be
implied that tridecanoic acid was produced by oxidation of α- partially hydrated by water, offering a plausible reason of α-
hydroxy myristic acid. α-Hydroxy myristic acid would have a keto acid formation in the α-oxidative decarboxylation of
chance to be further oxidised, because α-hydroxy myristic acid myristic acid.
also possesses the carboxylate group which accelerates the
Crystal structure analysis and MD simulation analysis
formation of Compound I of CYP152N1 using H2O2. In fact,
tridecanoic acid was produced when α-hydroxy myristic acid
To evaluate the enzymatically catalysed reaction mechanism
was used as a starting substrate (Fig. S8). α-Hydroxy myristic of α-keto acid formation reaction, we performed the structure
acid should be further oxidised to give α-keto myristic acid, analysis of substrate-bound form of CYP152N1. CYP152N1 was
whilst the α-keto fatty acid intermediate was not detected by crystallised by sitting drop vapour diffusion method. The
GC-MS. It was reported that various α-keto acids such as crystal structure of CYP152N1 was solved at 2.3 Å resolution
pyruvic acid26 and 4-methyl-2-oxopentanoic acid27 were (Fig. 4A, PDB: 5YHJ). The overall structure of CYP152N1
spontaneously decarboxylated by H2O2 under neutral or basic possessed a typical “P450 fold”31, 32 with a cysteine (Cys359)
conditions (Scheme 3)28, 29. We presume that α-keto myristic thiolate coordinated to the haem b cofactor (Fig. S11). A long
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electron density assignable to myristic acid was observed (Fig. throughout the entirety the 100 ns MD simulations (Fig. S13).
S12). The carboxylate of myristic acid interacts with Arg239 at The α-carbon atom of (S)-α-hydroxy myristic acid was closed to
the distal side of haem (Fig. 4B). The distance between the heme iron. These results lend further support to the enzymatic
haem iron and α-carbon atom of myristic acid was 5.2 Å, which oxidation of (S)-α-hydroxy myristic acid.
is shorter than that of the
β-carbon atom at 6.6 Å. This
observation is consistent with the α-selective hydroxylation of
myristic acid obtained in experiments. It is noteworthy to
mention here that Phe286 was observed at the corresponding
position to Phe288 in CYP152B1, which plays a critical role in
governing α-selective hydroxylation8, 33
.
Scheme 5 Possible reaction mechanism of α-keto myristic acid formation.
The possible catalytic reaction mechanisms of α-keto acids
formation reaction were shown in Scheme 5. The C-H
dissociation (Scheme 5A) and the O-H dissociation (Scheme 5B)
were possible to be catalysed by CYP152N1. However, the MD
simulation results supported the oxidation of (S)-α-hydroxy
myristic acid to α-keto myristic acid via the α-O-H cleavage
mechanism (Scheme 5B), since the (R)-α-hydrogen atom was
not shown to access the oxygen atom of Compound I during
the simulation time (Fig. 5).
Fig. 4 Whole structure (A) and active site structure (B) of myristic acid bound CYP152N1
(PDB: 5YHJ). Myristic acid, haem b, and amino residues are shown as sphere model or
stick model. Main chain of CYP152N1 is shown as ribbon model.
For the formation of α-keto myristic acid from α-hydroxy
myristic acid, the α-carbon atom of α-hydroxy myristic acid
needs to be near the haem iron in the same way to myristic
acid. To evaluate the binding of α-hydroxy myristic acid into
the active site of CYP152N1, we performed molecular
dynamics (MD) simulations of α-hydroxy myristic acid in the
CYP152N1 crystal structure. The initial model for α-hydroxy
myristic acid was prepared by transforming the pro-(S)-
hydrogen atom, which is locating at the α-position of myristic
acid in the CYP152N1 active site, into a hydroxy group. The oxo
Fig. 5 100ns distribution of distance from oxygen atom of compound I to hydrogen
atom of Compound I was placed at 1.65 Å perpendicular from atoms of (S)-α-hydroxy myristic acid. The plots of hydrogen atom of hydroxy myristic
acid at (S)-α-hydroxy group were shown as blue colour. The plots of (R)-α-hydrogen
the centre of the porphyrin ring. The carboxylate of (S)-α-
atom were shown as red colour. The plots of oxygen atom at (S)-α-hydroxy group were
hydroxy myristic acid remained hydrogen bonded to Arg239
shown as green colour.
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α-Oxidative Decarboxylation Reaction by Other CYP152s
relevant fatty acids such as stearic acid (C18), palmitic acid
(C16), myristic acid (C14), and these unsaturated fatty acids.
Further optimization of the reaction conditions is predicted to
enable control over both α-oxidative decarboxylation activities
and terminal-olefin productive decarboxylation activities of
CYP152 to generate ideal biofuels from recyclable biological
resources.
The α-oxidative decarboxylation reaction under high
concentration (1-10 mM) hydrogen peroxide was not reported
within 20 years from the discovery of CYP152s. Denning and
Faber et al. reported the myristic acid oxidation reaction of
CYP152L1 and CYP152A2 in the presence of 5 mM hydrogen
peroxide22, however, shorter chain fatty acids such as
tridecanoic acid and lauric acid were not detected.
Interestingly, CYP152B1, which is an α-selective fatty acid
peroxygenase, catalyses the α-oxidative decarboxylation
reaction of myristic acid at 5 mM hydrogen peroxide (Fig. 6B).
Experimental Section
Preparation and Purification of Enzymes
Moreover,
CYP152A1
catalyses
the
α-oxidative
Recombinant CYP152B1 possessing a GST-tag at its N-terminal was
expressed in Escherichia coli BL21 (DE3) [pGEX-AX2-CYP152B1] and
purified by GST affinity chromatography. Recombinant CYP152A1 with
a 6×Histidine-tag at its N-terminal was expressed in Escherichia coli
M15 [pQE30t-CYP152A1, pREP4] and purified via nickel chelate affinity
chromatography. GST-tag and 6×histidine-tag were removed from
both P450s by digestion with thrombin, followed by further
purification via gel filtration using BioAssist eZ according to a reported
procedure13. pQE30t-CYP152N1 (Fig. S1A) was designed from pQE30t-
CYP152A1 and the synthetic nucleic acid (pBlueScript II SK (+)-
CYP152N1) as described in the supporting information. About 18 mg/L
of CYP152N1 with 6xHis-tag at N-terminal were expressed in the
soluble fraction. CYP152N1 was purified in the same way to
CYP152A113. P450s in GB Buffer (100 mM potassium phosphate buffer
with 0.3 M potassium chloride and 20% glycerol) were flash frozen in
liquid nitrogen and stored at -80°C. The concentration of P450s was
calculated from the haem concentration determined via pyridine
decarboxylation reaction in concert with terminal-olefin-
productive decarboxylation reaction, α-hydroxylation reaction,
and β-hydroxylation (Fig. 6A).
ferro-hemochrome assay (εR,557 = 34.7 mM-1 cm-1)34, 35
.
Measurements
Fig. 6 GC-MS chart of myristic acid (C14) oxidation catalysed by CYP152A1 (A) and
CYP152B1 (B) in the presence of 5 mM H2O2. The TMS derivatized products were
analysed using a Shimadzu GCMS-QP2010 and DB-624 capillary column (30 m × 0.32
mm; Agilent Technologies, Inc., Santa Clara, CA). Retention time: 1-Tridecene (12.4
min), C12 (17.4 min), C13 (18.5 min), C14 (19.6 min), C13α-OH (20.4 min), C14α-OH (21.9
min), C14β-OH (22.1 min).
UV-visible spectra were recorded until no further absorbance change
occurred using a UV-2600 spectrophotometer (Shimadzu) at 25°C.
Spectra of ferric-CYP152N1 were recorded 6.2 μM under 100 mM
potassium phosphate buffer with 0.3 M potassium chloride and 20%
glycerol. Ferrous-CYP152N1 was prepared by the addition of sodium
dithionate to ferric-CYP152N1 solution. Spectra of ferric-CYP152N1 in
complex with substrate were recorded following the addition of
myristic acid, dissolved in 70% (v/v) EtOH/water solution. The
dissociation constant Kd was approximated from the absorbance
Conclusions
CYP152N1 catalysed the α-(S)-selective hydroxylation of
myristic acid in the presence of physiological concentrated
hydrogen peroxide. Crystal structure analysis supports the high
regio-selectivity of fatty acid oxidation. Following the addition
of excess hydrogen peroxide (> 5 mM), CYP152 family enzymes
switched to the production of shorter-chain fatty acid such as
tridecanoic acid as the main product. This α-oxidative
decarboxylation reaction consists of two distinct enzymatic
steps; 1) fatty acid α-hydroxylation, 2) α-keto acid formation,
followed by a third enzyme independent step; 3) α-keto acid
decarboxylation. It was reported that CYP152L1 catalyses the
olefin-productive (β-oxidative) decarboxylation reaction of
various chain-length fatty acids such as arachidic acid (C20) and
butanoic acid (C4). Although CYP152L1 (OleTJE) was regarded as
the best enzyme to produce biofuels as alkenes instead of bio-
alcohols and bio-diesels, the ideal chain length of biofuels for
industry or gasoline are shorter than the produced biologically
difference of the substrate free form by fitting with either
dissociation equation
a
.
Hydroxylation of Myristic Acid
The reaction cocktail contained 0.1 M potassium phosphate (pH 7.0),
0-250 μL of Myristic acid (C14), 50 nM of CYP152N1 and 200 μM of
H2O2 in a total volume of 1 mL. The reaction mixture was incubated at
37°C for 1 min, and thereafter the reaction was quenched with 2 mL of
dichloromethane followed by vigorous mixing. After the addition of ω-
hydroxy lauric acid (C12ω-OH) as an internal standard, the products
were extracted in dichloromethane. For derivatization of the extract,
25 μL of 0.01% (w/v) 9-anthryldiazomethan (ADAM®) acetone solution
was added, followed by incubation in the dark at room temperature
for 1 hr. Products were quantified by reverse phase HPLC analysis on a
Shimadzu SCL-10AVP systems equipped with Inertsil® ODS-3 column
according to a reported method8. The HPLC analytical conditions were
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as follows: flow rate 1.0 mL/ min; acetonitrile/water = 99/1; column concentrated to 16.4 mg/ml by centrifugation using Amicon Ultra
temperature, 30°C; extension wavelength, 365 nm, emission filters units (Millipore, Co.). The crustal of CYP152N1 was grown by the
wavelength 412 nm; and retention times, C12ω-OH (6.63 min), β- sitting-drop vapour diffusion using the reservoir solution 0.1 M Tris
hydroxy myristic acid (C14β-OH, 11.0 min), α-hydroxy myristic acid hydrochloride pH 8.5, 0.2 M magnesium chloride, 30% polyethylene
(C14α-OH, 12.3 min), C14 (22.2 min). To determine enantioselectivity of glycol 4,000 (A2 reagent of Pre-Crystallization Test, Hampton
the products, reverse phase HPLC analysis was performed using a Research). For the sitting-drop, 2 μL of CYP152N1 solution was mixed
Chiralpak AD-RH column installed on a Shimadzu SCL-10AVP systems. with 2 μL of the reservoir solution. The crystals of CYP152N1 for
The retention time was determined using arsenic samples. The diffraction experiment were dialyzed against the cryoprotectant
absolute configuration was assigned by the product ratios of the solution containing 25 mM MES 0.05 M Tris hydrochloride, 0.1 M
myristic acid hydroxylation catalysed by CYP152A113 and CYP152B16. magnesium chloride hexahydrate, 30% w/v polyethylene glycol 4,000
The HPLC analytical conditions were as follows: flow rate 0.9 mL/ min; and 10% glycerol for two days.
liner gradient MeOH/water = 85/15 (0-10 min) to 100/0 (100-120 min);
Data Collection and Refinement
column temperature, 40°C; extension wavelength, 365 nm, emission
wavelength 412 nm; and retention times, C14α-(R)-OH (33 min), C14α-
Crystals were flash-cooled in nitrogen gas stream at 100K. X-ray-
on beam
line BL26B1 at RIKEN SPring-8 (Hyogo, Japan) The program HKL200036
was used for the integration and scaling of the diffraction data. The
initial phase of the CYP152N1 crystal was obtained using MOLREP37
The atomic model was refined using REFMAC538, phenix.refine39, and
(S)-OH (36 min), C14β-(R)-OH (50 min), C14β-(S)-OH (53 min), C14 (57 diffraction data sets were collected at wavelength of 1.0
Å
min).
Oxidation of Myristic Acid under Elevated Concentration of
Hydrogen Peroxide
.
The reaction mixture contained 0.1 M potassium phosphate (pH 7.0), COOT40. TLS refinement41 was performed at the final stage of the
100 μM of Myristic acid (C14), 1 μM of CYP152N1 and 0.2-5 mM of H2O2 refinement by defining 8 separate TLS groups (chain A: 2-88, 89-226,
in a total volume of 1 mL. The reaction mixture was incubated at 37°C 227-377, 378-412, chain B: 2-88, 89-257, 258-377, 378-412). The
for 1 min, upon which the reaction was quenched with 1 mL of resulting models had a final Rwork of 21.4% and an Rfree of 24.8% (Table
dichloromethane followed by vigorous mixing. At the same time, 5 μL 1).
of 1 mM palmitic acid (C16) ethanol solution was added as the internal
Table 1 Data collection and refinement statistics
standard. Reaction products and internal standard were extracted in
Data collection
Space group
Refinement
Resolution
dichloromethane. For the derivatization of the extract, 25 μL of N,O-
bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% (v/v)
trimethylchlorosilane (TMCS) was added to 25 μL of dichloromethane
extract solution, and the mixture was incubated at room temperature
for more than 1 h. The derivatised products were analysed using a
Shimadzu GCMS-QP2010 and RxiTM-5ms capillary column (30 m × 0.25
mm; Restek Corp., Bellefonte, PA). The GC/MS analytical condition
were as follows: column temperature, 50°C (1 min) to 40°C/min (5
min) to 250°C (8 min); injection temperature, 250°C; interface
temperature 280°C; carrier gas, helium; flowrate, 0.9 ml/min; mode,
split mode; and split ratio, 1/50. the peaks of C14 (12.8 min), C14α-OH
(14.4 min), C14β-OH (14.6 min) and C13 (11.7 min) C12 (10.7 min) were
determined by the mass fragment patterns and authentic samples.
The peaks of C13α-OH were determined just by the mass fragment
patterns because authentic samples were not available. The
concentrations of substrate and products were determined by a
calibration curve of the ratio of products and internal standard peak
area.
P41
19.75-2.30
35736
Cell dimensions
No. reflections
a b c (Å)
Rwork/Rfree
58.96 58.96 238.34
0.2142/0.2478
Resolution
20.00-2.30
(2.38-2.30) *
No. of atoms
Protein
Rmerge
5.2 (33.0)
22.0 (5.1)
(0.981)
6686
118
61
I/σ(I)
Ligand
CC1/2
Water
Completeness
Redundancy
Wilson B (Å)
99.6 (100)
6.8 (6.7)
37.2
r.m.s deviations
Bond lengths (Å)
Bond angles (°)
0.009
1.1
*Values in parentheses are for the highest-resolution shell.; r.m.s.: root mean square
MD Simulation of Myristic Acid and αꢀHydroxy Myristic Acid
MD simulations were performed by using Maestro with
Desmond/GPU program42. The crystal structure of myristic acid binding
CYP152N1 was used as an initial template for MD simulations of
myristic acid. The template of α-hydroxy myristic acid was prepared by
exchanging the (S)-α-hydrogen atom with oxygen atom. The water
molecules present in the crystal structure were removed. Compound I
was modelled from the haem and the oxygen atom of the axial ligand
18O-Labelled Oxidant Reaction
The reaction mixture contained 0.1 M potassium phosphate (pH 7.0),
100 μM of Myristic acid (C14), 50 nM of CYP152N1 and 5 mM of 90%
18O labelled H2O2 in a total volume of 0.5 mL. The reaction mixture was
incubated at 37°C for 10 min, whereupon the reaction was quenched
by 1 mL of dichloromethane followed by vigorous mixing. At the same
time, the products were extracted by with dichloromethane. The
method of the GC/MS analyse was described above.
at 1.65
Å
above the Fe atom43, 44 . Hydrogen atoms were prepared by
the protein preparation wizard of Maestro. Protons of titratable
residue at pH 7.0 were estimated by PROPKA45, 46. All side chains of
glutamine, asparagine and histidine were not flipped, and all carboxyl
groups of glutamic acids and aspartic acids were deprotonated.
Histidine 94 was only double-protonated on the aromatic nitrogen
atoms. The proton of His208, His256, His363, and His408 were
prepared on δ-position, and that of His13, His92, His217, His319, and
Crystallisation of CYP152N1
The buffer for the purified CYP151N1 solution was exchanged to 50
mM MES buffer (pH 7.0) and 20% (v/v) glycerol. CYP152N1 was
6 | J. Name., 2012, 00, 1-3
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His357 were prepared on ε-position. The solvent box was prepared by CYP152N1
TIP4P47, 12 sodium cations were added to neutralise the total change,
Cytochrome
Exiguobacterium sp. AT1b (P450Exα
Cytochrome P450 isolated from Bacillus subtilis
IFO14144 (P450BSβ
Cytochrome P450 isolated from Clostridium
acetobutylicum ATCC824 (P450Cla
P450
isolated
from
)
and ions of 0.15 M sodium chloride were added to reduce the CYP152A1
interaction between hydrophilic residues at the protein surface using
System builder of Desmond48. The R.E.D. III derived atomic partial CYP152A2
charge of Compound I and thiolate ligand (Cys 359)49 were used for
)
)
our simulations. The protein back bone, Arg239 side chain, and CYP152A8
Compound I, except for the hydrogen atoms were restrained by the
Cytochrome P450 isolated from Alicyclobacillus
acidocaldarius LAA1 (CYP-Aa162)
force constant of 100 kcal (mol Å -1
)
to prepend braking of the CYP152B1
Cytochrome P450 isolated from Sphingomonas
CYP152N1 structure during the simulations. The simulation system was
equilibrated using the default protocol of Desmond. After a 5 ns CYP152L1
relaxation simulation, a 100 ns MD simulation was performed under
following conditions: equilibration of simulation was used isothermal CYP152L2
isobaric ensemble (NPT), forcefield was used as OPLS-200550, the
paucimobilis EY2395 (P450SPα)
Cytochrome P450 isolated from Jeotgalicoccus
sp. 8456 (OleTJE)
Cytochrome P450 isolated from Staphylococcus
massiliensis S46 (CYP-Sm46)
short-range electrostatic interactions between over 10
Å
divided CYP152P1
Cytochrome
P450
isolated
from
atoms were cut off, long-range electrostatic interactions were
computed with Particle Mesh Ewald method, all covalent bonds
involving hydrogen atoms were constrained using the SHAKE C16
algorism51, the thermostat and barostat were used as Nose-Hoover C14
chain52-54 at 300K and Martyna-Tobias-Kien55 at 1 atm with relaxation C13
of 1 ps and 2 ps, RESPA integrator56 was used with Fourier-space C12
electrostatics computed every 6 fs and all remaining interactions C14α-OH
computed every 2 fs. The result of MD simulation was analysed by C14β-OH
Methylobacterium populi sp. nov. (BJ001 T =ATCC
BAA-705 T =NCIMB 13946 T)57 (CYP-MP)
Palmitic acid (Hexadecanoic acid)
Myristic acid (Tetradecanoic acid)
Tridecanoic acid
Laulic acid (Dodecanoic acid)
α-Hydroxymyristic acid
β-Hydroxymyristic acid
Simulation Event Analysis of Desmond. Before the MD simulation of
α
C14α=O
α-Ketomyristic acid (α-Oxomyristic acid)
α-Hydroxytridecanoic acid
-hydroxy myristic acid, we checked the evaluation condition using 100 C13α-OH
ns MD simulation of myristic acid in the active site of CYP152N1. The
distance distribution between oxygen atom of compound
I and
1.
2.
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vitro selectivity (Fig. S4, α >99%, (S)-92%ee). 100 ns MD simulations of
α-hydroxy myristic acid bound CYP152N1 were performed in the same
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Acknowledgements
This work was supported, in part, by Grants-in-Aid-for Scientific
Research (B) to Y. W. (17H03087) and Grants-in-Aid-for Scientific
Research on Innovative Areas “Precise Formation of Catalyst Having
a Specified Field for Use in Extremely Difficult Substrate Conversion
Reactions” to O. S. (15H05806). from the Ministry of Education,
Culture, Sports, Science and Technology (Japan), and Grant-in-Aid
for JSPS Research Fellow to H. O. (16J02846). from Japan Society for
the Promotion of Science (Japan). We thank Dr. Go Ueno, Dr.
Hironori Murakami and Dr. Yuki Nakamura for assistance with the
data collection at SPring-8. We also thank Prof. David R. Nelson for
classification of CYP152 family enzymes.
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Notes and references
The atomic coordinates and structure factors (codes 5YHJ) have
been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
The abbreviations used are:
CYP or P450 Cytochrome P450
CYP152s
Cytochrome P450 152 family enzymes (Fatty
acid peroxygenases, EC 1. 11. 2. 4)
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