An unusual matrix of stereocomplementarity in the hydroxylation of monohydroxy fatty acids catalysed by cytochrome P450 from Bacillus megaterium with potential application in biotransformations

Article abstract of DOI:10.1039/a905974a

Cytochrome P₄₅₀ from Bacillus megaterium catalyses the diastereoselective hydroxylations of 13-hydroxymyristic acid, to predominantly erythro-12,13-dihydroxymyristic acid, and of 12-hydroxymyristic acid to give predominantly threo-12,13-dihydroxymyristic acid, in reactions that are stereocomplementary and with considerable potential application in biotransformations.

Full text of DOI:10.1039/a905974a

An unusual matrix of stereocomplementarity in the hydroxylation of
monohydroxy fatty acids catalysed by cytochrome P450 from Bacillus
megaterium with potential application in biotransformations
Farjad Ahmed,ac Eiman H. Al-Mutairi, Kathryn L. Avery, Paul M. Cullis,ac William U. Primrose,bc
d
ac
bc
Gordon C. K. Roberts and Christine L. Willisd
a
Department of Chemistry, University of Leicester, Leicester, UK LEI 7RH. E-mail: pmc@le.ac.uk
Department of Biochemistry, University of Leicester, Leicester, UK LEI 7RH
Centre for Mechanisms of Human Toxicity, University of Leicester, Leicester, UK LEI 7RH
School of Chemistry, University of Bristol, Cantock’s Close, Bristol, UK BS8 1TS
b
c
d
Received (in Liverpool, UK) 20th July 1999, Accepted 6th September 1999
Cytochrome P450 from Bacillus megaterium catalyses the
diastereoselective hydroxylations of 13-hydroxymyristic
acid, to predominantly erythro-12,13-dihydroxymyristic
acid, and of 12-hydroxymyristic acid to give predominantly
threo-12,13-dihydroxymyristic acid, in reactions that are
stereocomplementary and with considerable potential appli-
cation in biotransformations.
acids at the w-1, w-2 and w-3 positions, and also to give rise to
lesser amounts of dihydroxylated products.4a The production of
these dihydroxylated products clearly arises from a ‘second
round’ of hydroxylation of the first formed monohydroxy fatty
acids. The monohydroxy fatty acids are evidently poorer
substrates for cytochrome P450-BM3 (Table 1) such that the
amount of dihydroxylation product observed will depend on the
extent of reaction and the reaction conditions. Although there
has been a considerable amount of work reported on the
regioselectivity of monohydroxylation of fatty acids by cyto-
During the past decade, purified enzymes have been widely
used to effect transformations of value in synthetic chemistry.1
In addition to the familiar and widely exploited hydrolases and
reductases, there remains a considerable range of potentially
interesting and challenging enzyme-catalysed chemical trans-
formations, mostly notably the functionalisation of unactivated
positions, to be exploited in this context. We report here the
results of our studies on the stereoselectivity and regioselectiv-
ity shown by cytochrome P450 from Bacillus megaterium in the
hydroxylation of hydroxy fatty acids, and note a quite
remarkable matrix of stereoselective complementarity.
4a
chrome P450-BM3
,
hitherto there have been no studies on the
stereochemical courses of these processes. Furthermore, there
has been little reported on the hydroxylation of functionalised
fatty acids. In this study we have exploited the enantiomers of
12-hydroxy- 1 and (R)-13-hydroxy-myristic acid 2.
Cytochromes P450 form a diverse superfamily of enzymes
capable of inserting an atom of oxygen into a dazzling range of
substrates, e.g. fatty acids, prostaglandins, steroids, polycyclic
The route for the enantioselective synthesis of 12-hydroxy-
myristic acid is shown in Scheme 1. The enzyme-catalysed
reduction of 2-oxobutanoic acid with either lactate dehy-
2
aromatics. In contrast to the mammalian cytochromes P450
which are membrane-bound proteins, cytochromes P450 from
bacterial sources have proved more tractable for study at the
molecular level by virtue of being soluble proteins. The most
extensively studied of the bacterial enzyme systems is cyto-
chrome P450CAM from Pseudomonas which catalyses the
9
drogenase from Bacillus stearothermophilus (BS-LDH) or
hydroxyisocaproate dehydrogenase from Lactobacillus bulgar-
icus subsp. delbruckeii (LB-hicDH),10 gave, after esterification,
methyl 2-hydroxybutanoate (S)-3 and (R)-3 respectively. A
straightforward series of reactions led to the conversion of the
3
hydroxylation of camphor to the 5-exo-hydroxycamphor. This
system is a typical three component system requiring a
cytochrome P450 reductase, an iron–sulfur electron transfer
protein and the cytochrome P450 hydroxylase protein, and
exhibits a comparatively tight substrate specificity. Both of
these features detract from its use in biotransformations
although a number of mutant proteins of broader specificity
have recently been reported. In contrast, the class 2 cytochrome
hydroxy ester 3 to the C
6
-aldehyde 4 which, on reaction with the
C
8
-ylide, led to formation of predominantly the Z-alkene 5.
Finally deprotection of the alcohol and reduction of the double
bond gave either (S)- or (R)-12-hydroxymyristic acid 1. (R)-
13-Hydroxymyristic acid 2 was prepared by a similar route
from ethyl (R)-3-hydroxybutanoate.
In order to identify the products from the cytochrome P450
-
P
450 from Bacillus megaterium requires only a single redox
catalysed oxidation of 1 and 2, authentic samples of erythro-
and threo-12,13-dihydroxymyristic acids 6 and 7 were required.
component and this forms part of the multifunctional protein
that incorporates both the flavoprotein NADPH reductase
Treatment of acetaldehyde with the ylide derived from the C12
-
4
salt 8 gave a 9+1 mixture of Z+E alkenes 9 and 10 (Scheme 2).
domain and the heme-containing P450 domain. The enzyme has
been overexpressed and an X-ray crystal structure of the P450
domain has been determined. Cytochrome P450-BM3 catalyses
5
6
Table 1
the hydroxylation of a range of fatty acids to give mono- and di-
hydroxylated fatty acids. Capdevila et al. have reported the
Substrate
Myristate
K
M
/mM
k
cata/min21
7
selective oxidation of arachidonic acid, and our own prelimi-
8
3127
139
119
72
nary observations suggest that the range of potential alternative
substrates accepted by the enzyme may be quite large, including
(13R)-Hydroxymyristate
(12R)-Hydroxymyristate
(12S)-Hydroxymyristate
33
32
11
8
steroids, coumarins and sulfides. To determine its potential
utility in carrying out useful biotransformations we have
explored the regio- and stereo-selectivity in the P450-BM3
catalysed oxidation of hydroxylated fatty acids.
Wild-type cytochrome P450-BM3 is known to catalyse the
monohydroxylation of fatty acids such as myristic and palmitic
a
k
cat values are based upon monitoring of NADPH turnover, and with the
exception of myristate, where the coupling efficiency is known to be very
high, have not been corrected for uncoupled peroxidase for oxidase activity.
The values for hydroxymyristic acid are therefore upper estimates.
Chem. Commun., 1999, 2049–2050
This journal is © The Royal Society of Chemistry 1999
2049

this, starting from the (R)-13-hydroxymyristic acid, cytochrome
P
450-BM3 catalyses the second hydroxylation at the w-2 position
stereoselectivity to give predominantly the erythro-12,13-dihy-
droxymyrisitic acid [(12S,13R)-12,13-dihydroxymyristic acid,
8
3
5%; (12R,13R)-12,13-dihydroxymyristic acid 15%] (Scheme
). Clearly, in all three cases the preference is for oxygen
insertion into the C–H of the methylene group adjacent to the
hydroxymethylene moiety, rather than into the C–H of the
hydroxymethylene goups itself, which would lead to the
corresponding ketone. This would be consistent with a
mechanism that does not involve a hydrogen abstraction since
placement of the radical centre on the carbon atom bearing the
hydroxy group would have been expected to be energetically
preferred.
The observation of stereoselective hyroxylation of even
flexible long chain hydroxy fatty acids catalysed P450-BM3 has
demonstrated an interesting, stereocomplementary matrix of
biotransformations. The one major drawback that can be
envisaged is the requirement for NADPH, however we have
already addressed this by developing two systems in which the
reducing equivalents are provided directly from an electrode
Scheme 1 Reagents and conditions: i, BS-LDH, NADH, FDH, HCO
CH ; iii, LB-hicDH, NADH, FDH, HCO Na; iv, TBDMSOTf, pyridine;
v, DIBAL-H, toluene, 278 °C; vi, Ph PCHCO Et, CH CN; vii, H , Pd on
H Br , NaH, DMSO; xi, TBAF, THF.
2
Na; ii,
2
N
2
2
3
2
2
3
2
+
3 3 2 6 2
CaCO ; vii, Ph P CH(CH ) CO
1
3
thus allowing the oxidation to be driven electrochemically.
We have also demonstrated that P450-BM3 is not restricted to
long chain fatty acids and will tolerate a wide range of
functionality in the chain. This system holds great potential for
further exploitation in synthetic chemistry.
Notes and references
t
1 C. H. Wong, G. J. Shen, R. L. Pederson, Y. F. Wang and W. J. Hennen,
Methods Enzymol., 1991, 202, 591.
3 2 4
Scheme 2 Reagents: i, Bu OK, CH CHO; ii, I ; iii, OsO (cat), NMO.
2
Cytochrome P450: Structure Mechanism and Biochemistry, ed. P. R.
Ortiz de Montellano, 2nd edn., Plenum Press, New York, 1995.
Dihydroxylation of 9 using catalytic osmium tetroxide in the
presence of N-methylmorpholine N-oxide (NMO) gave the
erythro-diol 6. Isomerisation of the double bond with iodine
followed by dihydroxylation gave predominantly the threo-diol
3 T. L. Poulos, B. C. Finzel and A. J. Howard, J. Mol. Biol., 1987, 195,
687.
4 (a) Y. Miura and A. J. Fulco, Biochem. Biophys. Acta, 1975, 388, 305;
(b) L. O. Narhi and A. J. Fulco, J. Biol. Chem., 1986, 261, 7160.
7.
5
6
J. S. Miles, A. W. Munro, B. N. Rospendowski, W. E. Smith, J.
McKnight and A. J. Thompson, Biochem. J. 1992, 288, 503.
K. G. Ravichandran, S. S. Boddupalli, C. A. Hasemann, J. A. Peterson
and J. Deisenhofer, Science, 1993, 261, 731; H. Li and T. L. Poulos, Nat.
Struct. Biol., 1997, 4, 140.
Incubation of either enantiomer of 12-hydroxymyristic acid 1
or (R)-13-hydroxymyristic acid 2 with cytochrome P450-BM3
together with an NADPH-regenerating system11 (NADP,
2
glucose-6-phosphate, MgCl and glucose-6-phosphate dehy-
drogenase) both gave 12,13-dihydroxymyristic acid as a major
product (yields from 13R: 85%; 12R: 79%; 12S: 64% based on
total products identified by GC).12 Using the authentic samples
obtained above we have shown that hydroxylation of (R)-
7
J. H. Capdevila, S. Wei, C. Helvig, J. R. Flack, Y. Belosludtsev, G.
Truan, S. E. Graham-Lorence and J. A. Peterson, J. Biol. Chem., 1996,
271, 22663.
8 F. Ahmed, A. Celik, P. M. Cullis, W. U. Primrose and G. C. K. Roberts,
unpublished results.
9
1
2-hydroxymyristic acid catalysed by cytochrome P450-BM3 not
Full details of the synthetic route will be published later. For previous
biotransformations to give hydroxy acids, see D. Bur, M. A. Luyten, H.
Wynn, L. P. Provencher, J. B. Jones, M. Gold, J. D. Friesen, A. R.
Clarke and J. J. Holbrook, Can. J. Chem., 1989, 69, 1065; B. L.
Hirschbein and G. M. Whitesides, J. Am. Chem. Soc., 1982, 104, 4458;
M.-J. Kim and J. Y. Kim. J. Chem. Soc., Chem. Commun., 1991, 326.
only occurred regioselectivity but also stereoselectivity at w-1
giving predominantly the threo-12,13-dihydroxymyristic acid
[
1
1
(12S,13S)-12,13-dihydroxymyristic acid, 80%; (12S,13R)-
2,13-dihydroxymyristic acid 20%] (Scheme 3). Similarly (S)-
2-hydroxymyristic acid was regioselectivity and stereose-
lectively hydroxylated at the w-1 position to give predominantly
the enantiomeric threo-12,13-dihydroxymyristic acid diaster-
eoisomer [(12R,13R)-12,13-dihydroxymyristic acid, 80%;
10 N. Bernard, K. Johnsen, J. L. Gelpi, J. A. Alvarez, T. Ferain, D. Garmyn,
P. Hols, A. Cortes, A. R. Clarke, J. J. Holbrook and J. Delcour, Eur. J.
Biochem., 1997, 244, 213.
1
1 C. H. Wong and G. M. Whitesides, J. Am. Chem. Soc., 1981, 103,
890.
(12R,13S)-12,13-dihydroxymyristic acid 20%]. In contrast to
4
1
2 Enzymatic oxidation of fatty acids was performed by mixing cyto-
chrome P450 (30 mM), substrate (10 mM) and a NADPH-regenerating
system (comprising 10 mM NADP, 10 mM glucose-6-phosphate, 10
2
mM MgCl and 1 unit glucose-6-phosphate dehydrogenase) in phos-
phate buffer (15 ml, 0.1 M, pH 8.0). After incubation for 1 h, the
reactions were stopped by addition of 10 mL of 1 M HCl and the
products extracted with ethyl acetate (2 3 15 mL). GC-EIMS analysis
(70 eV) of the corresponding methyl ester was carried out on a Kratos
Concept mass spectrometer (Kratos Instruments. Inc) equipped with a
direct capillary interface to a Shimadzu 14A GC with electron impact
ionisation using a HP35 capillary GC column (30 m 3 0.25 mM HP35,
Hewlett Packard). Chromatography of the sample was performed at a
2
1
linear flow rate of helium gas of 68 cm s and, after 2 min at 180 °C,
2
1
the temperature was raised to 270 °C (at a rate 8 °C min ) and then held
at 270 °C.
1
3 F. Ahmed, A. P. Abbott, W. U. Primrose, P. M. Cullis and G. C. K.
Roberts, unpublished work, 1999, patent applied for.
Scheme 3 Reagents and conditions: i, P450 BM3, NADP, glucose-6-phos-
phate, MgCl
pH 8.0).
2
, glucose-6-phosphate dehyrogenase, phosphate buffer (0.1 M,
Communication 9/05974A
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Chem. Commun., 1999, 2049–2050