Enantioselective epoxidation of linolenic acid catalysed by cytochrome P450BM3 from Bacillus megaterium

Article abstract of DOI:10.1039/b506155e

Cytochrome P450_BM3, from Bacillus megaterium, catalyses the epoxidation of linolenic acid 1 yielding 15,16-epoxyoctadeca-9,12-dienoic acid 2 with complete regioand moderate enantio-selectivity (60% ee). The absolute configuration of the product is tentatively assigned as 15(R),16(S)-. The Michaelis-Menten parameters k_cat and K_m for the reaction were determined to be 3126 ± 226 min^-1 and 24 ± 6 μM respectively. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b506155e

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C O M M U N I C A T I O N
Enantioselective epoxidation of linolenic acid catalysed by
cytochrome P450BM3 from Bacillus megaterium†
Ayhan C¸ elik, Davide Sperandio, Robert E. Speight and Nicholas J. Turner*
School of Chemistry, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh,
UK EH9 3JJ. E-mail: n.j.turner@ed.ac.uk
Received 3rd May 2005, Accepted 23rd May 2005
First published as an Advance Article on the web 16th June 2005
Cytochrome P450BM3, from Bacillus megaterium, cataly-
ses the epoxidation of linolenic acid 1 yielding 15,16-
epoxyoctadeca-9,12-dienoic acid 2 with complete regio-
and moderate enantio-selectivity (60% ee). The absolute
configuration of the product is tentatively assigned as
15(R),16(S)-. The Michaelis–Menten parameters kcat and
Km for the reaction were determined to be 3126 226 min−1
and 24 6 lM respectively.
engineering methods to accept unnatural substrates.4 However,
the inherent complexity of cytochrome P450CAM involving a
multi-component system currently mitigates against its use as
a practical biocatalyst for organic synthesis.
In contrast, the soluble cytochrome P450 from Bacillus
megaterium (P450BM3) is catalytically self-sufficient, containing
both a haem-containing P450 domain and a cytochrome P450
reductase domain [Fig. 1(B)]. P450BM3 catalyses the hydrox-
ylation of saturated and unsaturated fatty acids along with
other substrates including alcohols, amides, etc.,5 with very
high turnover numbers and high stereoselectivity.6 Herein we
report the use of P450BM3 for the regio- and stereo-selective
epoxidation of linolenic acid 1 to produce epoxy-linolenic acid
2. The synthesis of oxygenated linolenic acid derivatives is of
interest in view of the fact that they possess bactericidal and/or
antifungal activities and play important roles in the defense
of various plants7 together with a variety of pathophysiologic
responses in plants,8 animals9 and even humans.10 In particular,
recent reports indicate that amongst the various fatty acids as
defense substrates, oxygenated linolenic acid showed significant
anti-rice fungus activities.11
Initial reactions were carried out on a small scale (2.8 mg of
linolenic acid 1) in the presence of a stoichiometric amount of the
co-factor, NADPH. Thereafter, larger scale reactions (70 mg of
linolenic acid), together with an NADPH-co-factor regenerating
system, were used to obtain sufficient amounts of product(s) for
full characterization. From the pilot experiments, samples were
taken at various time intervals and analyzed by LC–MS in order
to determine the time-course of the reaction and the product(s)
profile (Table 1). The reaction was found to proceed rapidly
and initially resulted in the production of a single oxygenated
(M + 16) product. However, when the reaction was allowed
to continue further, the formation of an additional metabolite
with mass of M + 32 was observed. Observations of this
kind are not unusual for P450-catalysed reactions as has been
previously noted12 in which high enzyme concentrations, long
incubation times, or/and low substrate concentrations can lead
to complex product profiles resulting from further oxygenation
of primary reaction products.
The use of enzymes and whole cells to carry out biotrans-
formations in synthetic chemistry has become well-established
in many laboratories over the past decade.1 In addition to
the familiar, and widely exploited, hydrolases and reductases,
there remains a considerable range of potentially interesting
and challenging enzyme-catalysed reactions. In particular, the
functionalisation of unactivated carbon atoms in an organic
molecule can be achieved by haem-containing enzymes, namely
cytochrome P450 monooxygenases. Cytochromes P450 are
capable of oxidizing a wide range of substrates, including
fatty acids, prostaglandins, steroids, polycyclic aromatics, etc.2
In contrast to the mammalian cytochromes P450s, which are
membrane-bound proteins, the corresponding enzymes from
bacterial sources have proved to be more practical for study at
the molecular level by virtue of being soluble proteins. The most
extensively studied enzyme in this class is cytochrome P450CAM
from Pseudomonas putida which catalyses the hydroxylation of
camphor to 5-exo-hydroxy camphor.3 This system is a typical
three-component protein arrangement requiring a cytochrome
P450 reductase, an iron–sulfur electron transfer protein and the
cytochrome P450 monooxygenase enzyme [Fig. 1(A)].
The results from the pilot-scale experiment were utilized for
the preparative-scale reaction, in which only one product with
an M + 16 mass was isolated together with recovery of some
starting material. When the reaction was allowed to proceed
further, four additional products, each with a mass of M +
32, were observed. The overall isolated yield for the mono-
oxygenated product was 64% with all other products, i.e. either
Fig. 1 (A) Three-component protein system, e.g. P450CAM; (B) catalyt-
Table 1 Time-dependent product profile for oxidation of linolenic acid
1 using P450BM3
ically self-sufficient P450BM3
.
Cytochrome P450CAM shows reasonably narrow substrate
specificity; nonetheless, there have been several examples re-
ported where its substrate specificity has been altered by protein-
Product distribution
Time/min
Conversion (%)
M + 16
M + 32
1
10
20
24
49
85
100
100
54
—
—
46
† Electronic supplementary information (ESI) available: detailed de-
scriptions of experimental procedures, analysis and characterization for
compound 2. See http://www.rsc.org/suppdata/ob/b5/b506155e/
2 6 8 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 8 8 – 2 6 9 0
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Table 2 Michaelis–Menten kinetic parameters of P450BM3-catalysed
di-oxygenated or di-hydroxylated products, accounting for a
further 25%. Further investigations did not resolve whether
these four products came from subsequent enzyme-catalysed
reactions or non-enzyme-catalysed epoxide ring opening under
the reaction conditions. Instead, attention was focussed on the
mono-oxygenated product. The reaction product with a mass
of M + 16 was conclusively identified as 15,16-epoxyoctadeca-
9,12-dienoic acid 2 based on the following data:
fatty acid oxidation
Entry
k
cat/min−1
Km/lM
kcat : Km/min−1 lM−1
Linolenic acid 1
3126 226 24
6
130
875
Eicosapentaenoic acid 1400 20 1.6 0.5a
Arachidonic acid
3200 40 1.2 0.1a 2666
a Given as spectral binding constant, Ks.
(i) Direct comparison of the 1H NMR spectra of the product
with that of linolenic acid revealed that the mono-epoxide had
been formed with the oxygen atom nearest the terminal position
of the linolenic acid chain. The triplet signal of the methyl
protons shifted from 0.99 ppm in linolenic acid 1 to 1.12 ppm in
the epoxide 2, due to the inductive effect of the adjacent oxygen
on the epoxy ring.
of oxidation must depend in some subtle way upon the way in
which the substrate binds at the active site and orients itself
towards oxidation.
The optical purity of the epoxide 2 obtained from P450BM3
-
catalysed oxidation of 1 was determined by initial conversion
to the methyl ester followed by HPLC analysis (Chiralcel OJ-
H, Diacel) using a racemic sample for comparison. HPLC
analysis indicated that the product had an enantiomeric excess
of 60%. The absolute configuration of 2 was tentatively assigned
as 15(R),16(S)-epoxyoctadeca-9,12-dienoic acid by direct com-
parison with published14 data for linoleic acid in the absence
of available data for linolenic acid. Thus it was assumed that
the order of elution of the 15(R),16(S)-enantiomer of linolenic
acid was the same as for linoleic, using the identical chiral
HPLC column, in which the methyl ester of 15(S),16(R)-
epoxyoctadeca-9,12-dienoic acid eluted first (9.2 min) followed
by 15(R),16(S)-epoxyoctadeca-9,12-dienoic acid (10.4 min).
The Michaelis–Menten kinetic parameters (kcat and Km) for
the oxidation of linolenic acid 1 were determined (Table 2)
using NADPH turnover rates and were compared with the
corresponding values for eicosapentaenoic acid and arachidonic
acid. The kcat of P450BM3 towards linolenic acid is comparable
to arachidonic acid, the best substrate known for the enzyme to
date.
In conclusion, we have shown that cytochrome P450BM3 can be
used for the preparative synthesis of enantiomerically enriched
epoxy-linolenic acid 2. Under the reaction conditions, the en-
zyme showed complete regio- and moderate enantio-selectivity
together with a very high turnover number, allowing the isolation
of epoxy-linolenic acid. This work, together with the report of
the oxidation of arachidonic acid,12 demonstrates the general
potential of cytochrome P450BM3 for selective oxidation of long
chain unsaturated fatty acids.
(ii) Co-elution and spectral comparison by GC–MS with au-
thentic standards which were prepared using chemical methods
(before carrying out the chemical epoxidation, linolenic acid
was converted to the methyl ester in methanol and catalytic
hydrochloric acid in order to analyse it by GC–MS). GC–
MS analyses revealed three mono-epoxy linolenic acids with
reasonably resolved peaks having retention times of 15.26, 15.30
and 15.34 min.
The distribution of products in cytochrome P450BM3-catalysed
oxidation of saturated and unsaturated fatty acids is mainly
dependent upon two factors, namely (i) the inherent reactivity
=
of the C–H/C C bonds, and (ii) the optimal orientation of
=
the C–H/C C bond with respect to the haem-bound reactive
oxygen intermediate. Hydroxylation of the saturated fatty acid
palmitic acid by cytochrome P450BM3 occurs preferentially at
the x-2 position along with substantial reaction at the x-1
and x-3 carbon atoms.13 In the case of unsaturated fatty acids
(Scheme 1) P450BM3 has been reported to catalyse both the
hydroxylation and epoxidation of arachidionic acid 3 yielding
18-hydroxyarachidonic acid and 14,15-epoxyarachidonic acid in
80 and 20% yields respectively).12 In contrast, with linolenic acid
we have observed exclusive epoxidation at the C-15,16-double
bond with no other detectable mono-hydroxylation products
under the experimental conditions. It is interesting to note that
similarly high levels of regioselectivity were observed in the
P450BM3-catalysed oxidation of linoleic acid14 although in this
case the exclusive product was the C-12,13-epoxy compound.
Thus for reasons that are not entirely clear, the site and nature
Acknowledgements
This work was supported by the Biological and Biotechnology
Science Research Council (A. C.). We also acknowledge the
Wellcome Trust for financial support and Dr Gareth Roberts
for his comments on the manuscript.
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Scheme 1 Product distribution from cytochrome P450BM3-catalysed
oxidation of arachidonic acid 3, linoleic acid 4, and linolenic acid 1.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 8 8 – 2 6 9 0
2 6 8 9

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2 6 9 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 6 8 8 – 2 6 9 0