Butane and propane oxidation by engineered cytochrome P450cam

Article abstract of DOI:10.1039/b110957j

The haem monooxygenase cytochrome P450_cam has been engineered to oxidise the gaseous alkanes butane and propane to butan-2-ol and propan-2-ol, respectively, by the use of bulky amino acid substitutions to reduce the volume of the substrate pocket and thus improve the enzyme-substrate fit: the F87W/Y96F/T101L/V247L mutant oxidises butane with a turnover rate of 750 min^-1 and 95% yield based on NADH consumed while the wild-type enzyme has an activity of 0.4 min^-1 with 4% yield.

Full text of DOI:10.1039/b110957j

Butane and propane oxidation by engineered cytochrome P450cam
Stephen G. Bell, Julie-Anne Stevenson, Helen D. Boyd, Sophie Campbell, Austin D. Riddle, Erica L.
Orton and Luet-Lok Wong*
Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road,
Oxford, UK OX1 3QR
Received (in Cambridge, UK) 29th November 2001, Accepted 18th January 2002
First published as an Advance Article on the web 7th February 2002
The haem monooxygenase cytochrome P450cam has been
engineered to oxidise the gaseous alkanes butane and
propane to butan-2-ol and propan-2-ol, respectively, by the
use of bulky amino acid substitutions to reduce the volume of
the substrate pocket and thus improve the enzyme–substrate
fit: the F87W/Y96F/T101L/V247L mutant oxidises butane
with a turnover rate of 750 min21 and 95% yield based on
NADH consumed while the wild-type enzyme has an
activity of 0.4 min21 with 4% yield.
haem by the threonine-101 ? leucine (T101L) mutation. The
butane and propane oxidation activity of wild-type P450cam and
the mutants Y96F, F87W/Y96F, Y96F/V247L, F87W/Y96F/
T101L, F87W/Y96F/V247L and F87W/Y96F/T101L/V247L
were determined.
In analysing the products of butane and propane oxidation,
we found that extraction with an organic solvent such as
chloroform,14 or solid-phase techniques9 were unsatisfactory.
The aqueous incubation mixtures were therefore injected
directly onto a SPB-1 column (0.5 mm ID 3 60 m). This
column tolerated water, and clearly resolved all the C1–C4
alcohols, aldehydes and ketones. We found that wild-type
P450cam and all the active site mutants oxidised butane to butan-
2-ol, and propane to propan-2-ol, with no evidence for the
formation of the terminal alcohols or the further oxidation
products butan-2-one and acetone.
The butane and propane oxidation activity and coupling
efficiency (defined as the percentage of NADH consumed that
lead to product formation, i.e. the yield based on NADH) for the
P450cam enzymes are given in Table 1. The wild-type enzyme
has low but detectable activity. As observed previously for
pentane and hexane,9 the Y96F mutant is two orders of
magnitude more active than the wild-type for butane and
propane oxidation. The data show that, in general, reducing the
active site volume by bulky amino acid substitutions increases
the alkane oxidation activity, consistent with our design
rationale. Butane oxidation is found to be faster than propane,
with both higher NADH turnover rates and coupling effi-
ciencies observed for the larger butane substrate. These trends
probably reflect the lower complementarity between the
mutants and the smaller propane molecule. We were surprised
by the high alkane oxidation activities conferred by just two
The selective oxidation of simple alkanes to alcohols under
ambient conditions, without further oxidation to aldehydes/
ketones or deep oxidation to carbon dioxide, is a reaction of
great industrial importance. The oxidation of methane to
methanol is of course a prime objective of catalysis research, but
oxidation of the other gaseous alkanes to the alcohols could also
afford new feedstocks for chemical synthesis. Solid state
heterogeneous catalysts require elevated temperatures and
pressures. On the other hand biological catalysts such as haem1
and non-haem monooxygenases2,3 catalyse alkane oxidation
under mild conditions and, as such, are potential candidates for
environmentally friendly, green chemistry alkane oxidation
catalysts.
Certain yeast strains can grow on long-chain (C8–C16) linear
alkanes as the only carbon and energy source by utilising the
haem-dependent cytochrome P450 monooxygenase for the
initial alkane oxidation step.4 However, the oxidation of
gaseous alkanes by any P450 enzyme has not been reported. We
have explored the protein engineering of cytochrome P450cam
5
from Pseudomonas putida for the oxidation of a range of
organic compounds,6–8 including pentane and hexane.9,10 We
proposed and showed that the specificity of P450cam could be
biased towards linear alkanes against the branched isomers by
reducing the active site volume with bulky amino acid
substitutions such as Val ? Leu.10 Here we report the further
engineering of P450cam for the oxidation of butane and
propane.
Our starting point for P450cam engineering is the Y96F/
V247L double mutant which favours the oxidation of hexane
over 3-methylpentane.10 We reasoned that butane and propane
oxidation could be promoted by further reducing the active site
volume to improve the enzyme–substrate fit and to constrain
these small molecules to bind closer to the haem. Substrate
binding close to the haem expels the water molecules in the
active site of substrate-free P450cam, which not only converts
the haem to high spin and thus give rise to faster rates of NADH
turnover but also suppresses the uncoupling pathway that forms
hydrogen peroxide.11,12 Substrate binding close to the haem
increases the probability of C–H bond oxidation by the ferryl
intermediate, thus suppressing the oxidase uncoupling path-
way.12
The active site structure of wild-type P450cam with bound
camphor is shown in Fig. 1.13 Both the Y96 and V247 side-
chains are located high in the active site pocket. In order to
constrain butane and propane close to the haem, we replaced
phenylalanine-87 with tryptophan (F87W) which has a ster-
ically more demanding indole side-chain. In addition, we
attempted to decrease the space available in the vicinity of the
Fig. 1 The active site structure of P450cam with bound camphor.
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CHEM. COMMUN., 2002, 490–491
This journal is © The Royal Society of Chemistry 2002

Table 1 The NADH oxidation and alcohol product formation rate and coupling efficiency of butane and propane oxidation catalysed by wild-type cytochrome
P450cam and active site mutants. Rates are given in nmol (nmol P450cam
21 (min)21. The coupling efficiency is defined as the proportion of NADH consumed
)
which is utilised by the enzyme for product formation and is given as a percentage. All values are means of at least 6 separate experiments, with all data within
15% of the respective means
Butane
Propane
NADH
oxidation ratea
Butan-2-ol
formation rateb
Coupling
efficiency
NADH
oxidation ratea
Propan-2-ol
formation rateb
Coupling
efficiency
Wild-type
Y96F
F87W/Y96F
Y96F/V247L
F87W/Y96F/T101L
F87W/Y96F/V247L
F87W/Y96F/T101L/V247L
10
100
200
250
540
263
795
0.4
42
132
186
460
88
4%
42%
66%
75%
85%
34%
95%
8
18
60
110
198
82
0.08
2.2
12
43
51
0.9%
12%
20%
39%
26%
17%
32%
13.7
110
750
346
a Incubations were carried out in cuvettes equipped with a screw cap and a Teflon septum. Mixtures (1.5 mL) contained 50 mM Tris pH 7.4, 1 µM P450cam
,
16 µM putidaredoxin, 1 µM putidaredoxin reductase, 200 mM KCl and 50 µg mL catalase. The mixture was oxygenated and the alkane substrate was added
as a saturated solution in 50 mM Tris, pH 7.4. The mixture was incubated at 30 °C for 2 min and NADH added to 400 µM by piercing the septum with a
syringe. The absorbance at 340 nm was monitored and the NADH turnover rate calculated using e340 = 6.22 mM21 cm21 b A 90 µL aliquot of an incubation
mixture was mixed with 10 µL of a 200 µM solution of the internal standard pentan-1-ol in water. The mixture was injected directly onto a SPB-1 gas
chromatography column. The column temperature was held at 40 °C for 4 min and then raised at 5 °C min21 to 100 °C. The retention times were: propan-2-ol,
4.35 min, butan-2-ol 6.53 min, pentan-1-ol 12.2 min.
mutations, viz. the F87W/Y96F and Y96F/V247L double
mutants. The very significant effects of the T101L mutation
most likely arise from both decreased active site volume and
increased active site hydrophobicity. Of the mutants studied in
this work the F87W/Y96F/T101L/V247L quadruple mutant has
the highest activity. The butane turnover rate of 750 min21
(2000 times faster than the wild-type enzyme) is quite
phenomenal because it is comparable to the camphor oxidation
rate by the wild-type enzyme (1200 min21) under identical
conditions, and the 95% coupling efficiency is no less
significant. Whilst the propane turnover rates are lower, it is
important to note that the activity trend for propane mirrors that
for butane, suggesting that high propane oxidation activity may
be achieved with additional mutations that further reduce the
active site volume.
Butane and propane are oxidised exclusively at the secondary
C–H bonds. The absence of attack at the primary C–H bonds
reflects substrate binding orientation and mobility which allow
the ferryl intermediate to select the more activated secondary
C–H bonds.9 Molecular dynamics simulations of camphor
oxidation by wild-type P450cam suggested that the camphor C5
and C9 are at the same distance from the ferryl oxygen but only
the more activated, secondary C–H bond at C5 is attacked.15
Hence primary C–H bond oxidation would only be observed if
the substrate is constrained such that only primary C–H bonds
are close to the haem.
mutants studied here, but equally we expect the activity to
increase when other appropriate mutations are introduced. The
exciting prospect remains of engineering P450cam into a
methane monooxygenase.
Notes and references
1 Cytochrome P450: Structure, Mechanism, and Biochemistry, ed. P. R.
Ortiz de Montellano, Plenum Press, New York, 1995.
2 H. Dalton, P. Wilkins and Y. Jiang Structure and mechanism of action
of the hydroxylase of soluble methane monooxygenaseA, in AMicrobial
growth on C1 compounds, ed. J. C. Murrell and D. P. Kelly, Intercept
Press, Andover, UK, 1993, pp. 65.
3 J. D. Lipscomb, Annu. Rev. Microbiol., 1994, 48, 371.
4 J. G. Leahy and R. R. Colwell, Microbiol. Rev., 1990, 54, 305.
5 I. C. Gunsalus and G. C. Wagner, Methods Enzymol., 1978, 52, 166.
6 L. L. Wong, A. C. G. Westlake and D. P. Nickerson, Struct. Bonding,
1997, 88, 175.
7 J. P. Jones, E. J. OAHare and L. L. Wong, Chem. Commun., 2000,
247.
8 S. G. Bell, R. J. Sowden and L. L. Wong, Chem. Commun., 2001,
635.
9 J.-A. Stevenson, A. C. G. Westlake, C. Whittock and L.-L. Wong, J. Am.
Chem. Soc., 1996, 118, 12846.
10 J.-A. Stevenson, J. K. Bearpark and L.-L. Wong, New J. Chem., 1998,
551.
11 E. J. Mueller, P. J. Loida and S. G. Sligar ‘Twenty-five years of P450cam
research. Mechanistic insights into oxygenase catalysis’, in ACyto-
chrome P450: Structure, Mechanism, and BiochemistryA, ed. P. R. Ortiz
de Montellano, Plenum Press, New York, 1995, pp. 83.
12 P. J. Loida and S. G. Sligar, Biochemistry, 1993, 32, 11530.
13 T. L. Poulos, B. C. Finzel and A. J. Howard, J. Mol. Biol., 1987, 195,
687.
14 D. P. Nickerson, C. F. Harford-Cross, S. R. Fulcher and L. L. Wong,
FEBS Lett., 1997, 405, 153.
15 M. D. Paulsen and R. L. Ornstein, Proteins Struct. Funct. Genet., 1991,
11, 184.
In conclusion we have used rational protein engineering to
generate, for the first time, a P450 enzyme capable of rapid and
efficient oxidation of gaseous alkanes. We also attempted
ethane and methane oxidation with the mutants but the activity
was barely detectable by gas chromatography. We note from the
data in Table 1 that the alkane oxidation activity for each mutant
dropped by approximately an order of magnitude from butane to
propane. It is likely then that the ethane oxidation activity will
be at least an order of magnitude lower than for propane for the
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