Molecular recognition in (+)-α-pinene oxidation by cytochrome P450cam

Article abstract of DOI:10.1021/ja028460a

Oxygenated derivatives of the monoterpene (+)-α-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-α-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450_cam from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450_cam for the selective oxidation of (+)-α-pinene. Consideration of the structures of (+)-camphor and (+)-α-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-α-pinene. Some mutants had tighter (+)-α-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-α-pinene oxidation rate of 270 nmol (nmol of P450_cam)^-1 min^-1, which was 70% of the rate of camphor oxidation by wild-type P450_cam. Camphor is oxidized by wild-type P450_cam exclusively to 5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-α-pinene occupied similar positions to those found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450_cam enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-α-pinene epoxides were among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of the P450_cam mutants initially examined, was determined to provide further insight into P450_cam substrate binding and catalysis. (+)-α-Pinene was bound in two orientations which were related by rotation of the molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product distribution. In particular competition rather than cooperativity between the F87W and V247L mutations and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450_cam mutant that could oxidize (+)-α-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic oxidation of (+)-α-pinene, which is readily available in large quantities, could have applications in synthesis. The results also show that the protein engineering of P450_cam for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme - substrate interactions.

Full text of DOI:10.1021/ja028460a

Published on Web 12/21/2002
Molecular Recognition in (+)-r-Pinene Oxidation by
Cytochrome P450cam
Stephen G. Bell,† Xuehui Chen,‡ Rebecca J. Sowden,† Feng Xu,‡
Jennifer N. Williams,† Luet-Lok Wong,*,† and Zihe Rao*,‡
Contribution from the Department of Chemistry, Inorganic Chemistry Laboratory, UniVersity of
Oxford, South Parks Road, Oxford, OX1 3QR, U.K., and Laboratory of Structural Biology,
School of Life Sciences and Engineering, Tsinghua UniVersity, Beijing 100084, China
Received September 8, 2002; E-mail: raoz@xtal.tsinghua.edu.cn; luet.wong@chem.ox.ac.uk
Abstract: Oxygenated derivatives of the monoterpene (+)-R-pinene are found in plant essential oils and
used as fragrances and flavorings. (+)-R-Pinene is structurally related to (+)-camphor, the natural substrate
of the heme monooxygenase cytochrome P450cam from Pseudomonas putida. The aim of the present work
was to apply the current understanding of P450 substrate binding and catalysis to engineer P450cam for
the selective oxidation of (+)-R-pinene. Consideration of the structures of (+)-camphor and (+)-R-pinene
lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations.
All mutants showed greatly enhanced binding and rate of oxidation of (+)-R-pinene. Some mutants had
tighter (+)-R-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L
-1
mutant, with a (+)-R-pinene oxidation rate of 270 nmol (nmol of P450cam
)
min-1, which was 70% of the
rate of camphor oxidation by wild-type P450cam. Camphor is oxidized by wild-type P450cam exclusively to
5-exo-hydroxycamphor. If the gem dimethyl groups of (+)-R-pinene occupied similar positions to those
found for camphor in the wild-type structure, (+)-cis-verbenol would be the dominant product. All P450cam
enzymes studied gave (+)-cis-verbenol as the major product but with much reduced selectivity compared
to camphor oxidation by the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-R-pinene epoxides were
among the minor products. The crystal structure of the Y96F/F87W/V247L mutant, the most selective of
the P450cam mutants initially examined, was determined to provide further insight into P450cam substrate
binding and catalysis. (+)-R-Pinene was bound in two orientations which were related by rotation of the
molecule. One orientation was similar to that of camphor in the wild-type enzyme while the other was
significantly different. Analysis of the enzyme/substrate contacts suggested rationalizations of the product
distribution. In particular competition rather than cooperativity between the F87W and V247L mutations
and substrate movement during catalysis were proposed to be major factors. The crystal structure lead to
the introduction of the L244A mutation to increase the selectivity of pinene oxidation by further biasing the
binding orientation toward that of camphor in the wild-type structure. The F87W/Y96F/L244A mutant gave
86% (+)-cis-verbenol and 5% (+)-verbenone. The Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol
but interestingly also 32% (+)-verbenone, suggesting that it may be possible to engineer a P450cam mutant
that could oxidize (+)-R-pinene directly to (+)-verbenone. Verbenol, verbenone, and myrtenol are naturally
occurring plant fragrance and flavorings. The preparation of these compounds by selective enzymatic
oxidation of (+)-R-pinene, which is readily available in large quantities, could have applications in synthesis.
The results also show that the protein engineering of P450cam for high selectivity of substrate oxidation is
more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of
enzyme-substrate interactions.
Introduction
of largest class of organic compounds found in biological
systems. Monoterpenes (n ) 2) are more volatile than their
sesquiterpenoid (n ) 3) homologues. The involatile diterpenes
such as squalene (n ) 4) and larger terpenoids have important
biological activities; e.g., some are hormones or precursors to
hormones.
Terpenes have the general formula (C5H8)n and are biosyn-
thesized from isoprene units in the form of isopentenyl
pyrophosphate.1 After the head-to-head and head-to-tail coupling
of isoprene units, a series of carbonium ion rearrangements leads
to a wide variety of cyclic and acyclic terpene skeletal
structures.The parent terpenes and their oxidation products, such
as the epoxides, alcohols, aldehydes, and ketones, constitute one
Many mono- and sesquiterpenoid compounds found naturally
in plant essential oils are sought-after fragrances and flavorings
due to their distinctive, pleasant odors and taste notes.2,3 For
example (-)-menthol, which gives peppermint the characteristic
cooling sensation, (-)-carvone, which gives the spearmint
† University of Oxford.
‡ Tsinghua University.
(1) Gershenzon, J.; Croteau, R. B. Lipid Metab. Plants 1993, 339-388.
9
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J. AM. CHEM. SOC. 2003, 125, 705-714
705

A R T I C L E S
Bell et al.
flavor, and (-)-perillyl aldehyde, found in the herb perilla, are
derived from the monoterpene (-)-limonene. The highly specific
cytochrome P450 monooxygenases (CYP71D18 from spearmint
and CYP71D13 from peppermint), which catalyze the initial
oxidation of (-)-limonene in the respective plants, have been
expressed and their properties investigated extensively.4-9
Verbenol, verbenone, and myrtenol are derived from the
oxidation of R-pinene. However, the enzymes involved in
biological R-pinene oxidation are not yet well characterized.10-12
The desirable properties of monooxygenated terpenoids meant
that they are high added-value fine chemicals. The one-step or
one-pot synthesis of these compounds by direct oxidation of
the readily available parent terpenes could therefore have
applications in synthesis.
Cytochrome P450cam (CYP101)13,14 from the soil bacterium
Pseudomonas putida catalyzes the oxidation of (+)-camphor
to 5-exo-hydroxycamphor, the first step in the camphor me-
tabolism pathway of the organism.15,16 The P450cam system has
been studied extensively,17 and the crystal structure of different
forms of the enzyme with a variety of molecules bound within
the active site have been determined.18-29 The bicyclic com-
pound (+)-R-pinene is structurally related to camphor (Figure
1). We are interested in the possibility of applying the current
knowledge of P450cam substrate binding and recognition,
Figure 1. Molecular structures of (+)-camphor, (+)-camphane, and (+)-
R-pinene.
mechanism, and dynamics to engineer the enzyme for the
selective oxidation of (+)-R-pinene. Such engineered variants
may have applications in synthesis and also could provide a
means for discovering compounds with novel biological proper-
ties.
In the present study, we designed a number of active-site
mutations based on a comparison of the structures of camphor
and (+)-R-pinene. The activity, selectivity, and coupling ef-
ficiency of these P450cam mutants for (+)-R-pinene oxidation
were assayed. We also determined the crystal structure of the
F87W/Y96F/V247L triple mutant with (+)-R-pinene bound
within the active site. Based on the enzyme/substrate contacts
revealed by the structure, and the results of the activity studies,
further mutations were carried out and improved selectivity for
the formation of (+)-cis-verbenol was obtained.
(2) Arctander, S. Perfume and FlaVor Materials of Natural Origin; Allured
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(3) Charlwood, B. V.; Charlwood, K. A. Methods Plant Biochem. 1991, 7,
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1990, 276, 219-226.
Materials and Methods
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2000, 379, 127-136.
The atomic coordinates have been deposited in the Protein Data
Bank, code 1MPW.
(6) Wust, M.; Little, D. B.; Schalk, M.; Croteau, R. Arch. Biochem. Biophys.
2001, 387, 125-136.
(7) Wust, M.; Croteau, R. B. Biochemistry 2002, 41, 1820-1827.
(8) Schalk, M.; Croteau, R. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 11 948-
11 953.
General Materials. Enzymes for molecular biology were from New
England Biolabs. Buffer components were from Anachem Ltd. General
reagents were from Sigma-Aldrich or Merck. NADH was from Roche
Diagnostics. (+)-R-Pinene, R-pinene epoxide, (-)-cis-verbenol, (+)-
myrtenol, (+)-perillyl alcohol, and (-)-carvone were from
Sigma-Aldrich. UV/vis spectra were recorded on a CARY 1E double-
beam spectrophotometer equipped with a Peltier cell temperature
controller ((0.1 °C). Gas chromatographic (GC) analyses were
performed on a Fisons 8000 series instrument equipped with a flame-
ionization detector (FID). Two columns were used, a DB-1 fused silica
capillary column (0.25 mm i.d. × 30 m) for general analysis and a
â-DEX chiral phase column (0.5 mm i.d. × 30 m) for the separation
of optical isomers.
Enzymes and Molecular Biology. General DNA and microbiologi-
cal manipulations were carried out by standard methods.30 Cytochrome
P450cam and its associated electron-transfer proteins putidaredoxin and
putidaredoxin reductase were expressed in Escherichia coli and purified
following standard methods.31-33 The purified proteins were stored at
-20 °C in buffers containing 50% glycerol. Immediately before use
the proteins were buffer exchanged into 50 mM Tris, pH 7.4, on a
5-mL bed volume PD-10 gel filtration column (Amersham Biosciences).
Site-directed mutagenesis was carried out by using the Stratagene
QuikChange mutagenesis kit with oligonucleotide primers designed
following the manufacturer’s guidelines. Some mutants were prepared
by cloning fragments of the P450cam gene from different mutants using
(9) Lupien, S.; Karp, F.; Wildung, M.; Croteau, R. Arch. Biochem. Biophys.
1999, 368, 181-192.
(10) Colocousi, A.; Leak, D. J. EnViron. Biotechnol. 1996, 144-157.
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341.
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R.; Fujii-Kuriyama, Y.; Gonzalez, F. J.; Guengerich, F. P.; Gunsalus, I.
C.; Johnson, E. F.; Loper, J. C.; Sato, R.; Waterman, M. R.; Waxman, D.
J. DNA Cell Biol. 1991, 10, 1-14.
(14) Nelson, D. R.; Koymans, L.; Kamataki, T.; Stegeman, J. J.; Feyereisen,
R.; Waxman, D. J.; Waterman, M. R.; Gotoh, O.; Coon, M. J.; Estabrook,
R. W.; Gunsalus, I. C.; Nerbert, D. W. Pharmacogenetics 1996, 6, 1-42.
(15) Gunsalus, I. C.; Wagner, G. C. Methods Enzymol. 1978, 52, 166-188.
(16) Sligar, S. G.; Murray, R. I. In Cytochrome P450: Structure, Mechanism,
and Biochemistry, 1st ed.; Ortiz de Montellano, P. R., Ed.; Plenum Press:
New York, 1986; pp 429-503.
(17) Mueller, E. J.; Loida, P. J.; Sligar, S. G. In Cytochrome P450: Structure,
Mechanism, and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.;
Plenum Press: New York, 1995; pp 83-124.
(18) Poulos, T. L.; Finzel, B. C.; Gunsalus, I. C.; Wagner, G. C.; Kraut, J. J.
Biol. Chem. 1985, 260, 16 122-16 130.
(19) Poulos, T. L.; Finzel, B. C.; Howard, A. J. Biochemistry 1986, 25, 5314-
5322.
(20) Poulos, T. L.; Finzel, B. C.; Howard, A. J. J. Mol. Biol. 1987, 195, 687-
700.
(21) Raag, R.; Poulos, T. L. Biochemistry 1989, 28, 7586-7592.
(22) Raag, R.; Poulos, T. L. Biochemistry 1991, 30, 2674-2684.
(23) Raag, R.; Poulos, T. L. Biochemistry 1989, 28, 917-922.
(24) Vidakovic, M.; Sligar, S. G.; Li, H.; Poulos, T. L. Biochemistry 1998, 37,
9211-9219.
(25) Raag, R.; Martinis, S. A.; Sligar, S. G.; Poulos, T. L. Biochemistry 1991,
30, 11420-11429.
(26) Schlichting, I.; Jung, C.; Schulze, H. FEBS Lett. 1997, 415, 253-257.
(27) Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A. M.; Maves, S. A.; Benson,
D. E.; Sweet, R. M.; Ringe, D.; Petsko, G. A.; Sligar, S. G. Science 2000,
287, 1615-1622.
(30) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory
Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: New York, 1989.
(31) Westlake, A. C.; Harford-Cross, C. F.; Donovan, J.; Wong, L. L. Eur. J.
Biochem. 1999, 265, 929-935.
(28) Di Gleria, K.; Nickerson, D. P.; Hill, H. A. O.; Wong, L.-L.; Fu¨lo¨p, V. J.
Am. Chem. Soc. 1998, 120, 46-52.
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6066-6073.
(29) Li, H.; Narasimhulu, S.; Havran, L. M.; Winkler, J. D.; Poulos, T. L. J.
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9
706 J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003

Molecular Recognition in (+)-R-Pinene Oxidation
A R T I C L E S
Table 1. X-ray Data Collection, Refinement, and Common
the unique Nde I restriction site at the 5′-end of the gene, the internal
Sph I site which spanned amino acids 121-123, and the Hind III site
at the 3′-end of the gene. Thus the Y96F/ L224A/V247L mutant was
generated by ligating together the Nde I/Sph I fragment of the Y96F
mutant (which contained the Y96F mutation) with the Sph I/Hind III
fragment of the F87W/Y96F/L224A/V247L mutant (containing the
L244A and V247L mutations). Mutants were identified and fully
sequenced by automated DNA sequencing on an ABI 377XL Prism
DNA sequencer by the DNA sequencing facility at the Department of
Biochemistry, University of Oxford. All P450cam enzymes described
in this work also contained the C334A mutation to prevent protein
dimerization via disulfide bond formation.34 For convenience, the
C334A base mutant is referred to as “wild-type”, the triple mutant
F87W/Y96F/C334A as F87W/Y96F, etc.
Structural Parameters
data
unit cell
a ) 66.90 Å, b ) 62.52 Å, c ) 95.70 Å
â ) 90.3°
P21
space group
no. of molecules in unit cell
refinement
2 (A,B)
502.3 Å
300 428
33 002
total no. of reflections
unique reflections
completeness (highest
resolution shell)
(I)/[σ(I)]
97.1% (84.3%)
20
8.6%
19.4%
26%
a
Rmerge
b
Rwork
b
Rfree
P450cam Crystallization and Substrate Soaking Experiments.
Crystals of the F87W/Y96F/V247L mutant were grown at 291 K by
the vapor diffusion method. Immediately prior to crystallization the
mutant was further purified by size-exclusion chromatography on a
Superdex 75 (Amersham Biosciences) column (26 mm i.d. × 80 cm)
by eluting with 40 mM phosphate, pH 7.4, containing 1 mM camphor,
10 mM â-mercaptoethanol, and 200 mM KCl. The column was eluted
at a flow rate of 0.8 mL min-1, and orange-red P450cam fractions with
residues not modeled
no. of molecules with
(+)-R-pinene bound
rmsd from restraint values
A1-A9, B1-B9
2 (A,B)
bond lengths 0.0016 Å
bond angles 1.76°
WT vs A 0.39 Å; WT vs B 0.44 Å
rmsd between CR backbone
of molecules
A vs B 0.36 Å
Fe-S (cysteine 357) distance A 2.29 Å; B 2.08 Å; WT 2.20 Å
A404/A280 g 1.1 were pooled and used for crystallization. The protein
Fe-porphyrin plane distance
Ramachandran analysis:
most favored
A 0.36 Å; B 0.36 Å; WT 0.37 Å
A 88.2% (11.8%); B 84.8% (15.2%)
was buffer exchanged into 100 mM cacodylate, pH 6.5, and 200 mM
KCl and concentrated to 8 mg mL-1 by ultrafiltration. A 1-µL aliquot
of this solution was mixed with 1 µL of 30% PEG8000 in buffer A
(100 mM cacodylate, pH 6.5, 200 mM sodium acetate) and suspended
over 1 mL of 18-24% PEG8000 in buffer A. Diffraction quality
crystals of P450cam appeared within 24 h. Crystals were captured with
a fiber loop and soaked in 100 mM MES, pH 6.5, 100 mM sodium
acetate, 100 mM KCl, and 20% PEG8000 containing 100 µM (+)-R-
pinene (added as a 100 mM stock in ethanol) for 5 days.
(additionally allowed)
a Rmerge ) Σ|(I) - I|/Σ(I) over all reflections. Rwork ) Σ|Fo - Fc|/Fo
for all reflections, Rfree calculated from against 8% reflections withheld at
random.
b
refinement parameters were Rwork ) 19.4% and Rfree ) 26.0%. The
data collection and structure refinement statistics are summarized in
Table 1.
Data Collection and Structure Refinement. Immediately prior to
data collection, crystals were soaked in a cryoprotecting solution
consisting of 100 mM MES, pH 6.5, 100 mM KCl, 100 mM sodium
acetate, 20% glycerol, and 20% PEG8000 and flash frozen at 100 K in
a stream of cold nitrogen gas. X-ray diffraction data were collected at
100 K on a MAR345 image plate by using Cu KR radiation (λ ) 1.5418
Å) from an in-house Rigaku rotating anode X-ray generator operating
at 48 kV and 98 mA. The crystals belonged to the space group P21
with unit cell dimensions a ) 66.90 Å, b ) 62.52 Å, c ) 95.70 Å, and
â ) 90.28°. A total of 300 428 reflections were measured, with a Rmerge
of 8.6% for 33 002 unique reflections and 97.1% completeness (50-
2.3 Å). Data were collected to 84.3% completeness in the highest
resolution shell.
Substrate Binding Constant and NADH Turnover Rate Deter-
minations. All (+)-R-pinene binding and oxidation assays were carried
out at 30 °C. The substrate-free form of the P450cam enzymes, obtained
by passage through a PD-10 gel filtration column, were diluted to 1
µM. KCl was added from a 2 M stock in 50 mM Tris, pH 7.4 buffer,
to 200 mM. The substrate was added from ethanolic stocks and the
spectra of the mixed high-spin/low-spin form of the enzyme at different
pinene concentrations were recorded. Ethanol on its own at the final
total concentration of ca. 5% did not induce any change in the spectra.
The (+)-R-pinene binding constant was determined according to
literature methods.35
Incubation mixtures for NADH turnover assays (1.5 mL) contained
50 mM Tris, pH 7.4, 200 mM KCl, 1 µM P450cam, 4 µM putidaredoxin,
1 µM putidaredoxin reductase, and 30 µg µL-1 bovine liver catalase.36
The mixtures were oxygenated and then equilibrated at 30 °C for 2
min. The (+)-R-pinene substrate was added as a 100 mM stock in
ethanol to a final concentration of 400 µM. NADH was added to 250
µM and the absorbance at 340 nm was monitored. The reaction mixtures
were stirred throughout the time-course of the reaction. The maximum
concentration of ethanol in an incubation reaction was 0.4% v/v, and
control experiments showed that ethanol at concentrations up to 10%
v/v did not induce any increase in NADH consumption above
background.
The structure was solved by molecular replacement based on the
crystal structure of wild-type P450cam with bound camphor (protein
databank code: 2CPP). In the case of the mutant crystal, although the
three angles of the unit cell were all virtually 90°, Rsym in the
orthorhombic system was considerably higher than for a monoclinic
cell, especially in higher resolution shells. More importantly, extensive
molecular replacement was attempted in all possible orthorhombic space
groups without any success. Hence the mutant was determined to be
of P21 space group for which we were able to find two molecular
replacement solutions corresponding to the two molecules per asym-
metric unit. The P21 space group was also found to be the case in a
similar study.27 The space group parameters suggested that the crystals
have 222 pseudo-symmetry and this was further confirmed by inspection
of the crystal packing. The two molecules in the same asymmetric unit
were 62 Å apart with a rotation angle of 13.5° along an axis
perpendicular to the z axis and at 97° to the x axis when superimposed.
As evident in the difference Fourier map, both molecules showed clear
electron density above the heme group, which could be modeled as
(+)-R-pinene but with different substrate orientations. The final
Product Formation Rate Determinations. After all the NADH had
been consumed in an incubation experiment, 1 mL of the mixture was
transferred to a glass centrifuge tube. (-)-Carvone was added from a
10 mM stock in ethanol to 50 µM final concentration to act as an
internal standard for the extraction and GC analysis steps. The organics
were extracted by vortexing with 250 µL of prechilled (4 °C)
chloroform. The phases were separated by centrifuging at 3500g,
(35) Peterson, J. A. Arch. Biochem. Biophys. 1971, 144, 678-693.
(36) Stevenson, J.-A.; Westlake, A. C. G.; Whittock, C.; Wong, L.-L. J. Am.
Chem. Soc. 1996, 118, 12846-12847.
(34) Nickerson, D. P.; Wong, L.-L. Protein Eng. 1997, 10, 1357-1361.
9
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A R T I C L E S
Bell et al.
4 °C, and the chloroform layer was carefully removed. The samples
were either analyzed immediately or stored at -20 °C until required.
The organic extracts were analyzed by GC with the flame ionization
detector set at 250 °C and the injector at 150 °C. The temperature of
the DB-1 column was held at 120 °C. The retention times were as
follows: pinene epoxides, 4.65 and 4.80 min; (+)-cis-verbenol, 5.25
min; (+)-myrtenol, 6.30 min; (+)-verbenone, 6.40 min. The DB-1
column did not have a chiral stationary phase and so would not separate
the enantiomers of a chiral compound, e.g., (+)-cis-verbenol and (-)-
cis-verbenol would have identical retention times, but diastereoisomers
such as (+)-cis-verbenol and (+)-trans-verbenol were resolved. The
chiral phase â-DEX column, on the other hand, resolved all isomers.
The concentration of (+)-R-pinene oxidation products in an incuba-
tion mixture was determined by calibration of the FID response for
the products using (+)-perillyl alcohol as a representative example of
oxygenated pinenes. The assumption was that these isomeric compounds
would have near identical responses on the FID, and this was shown
to be the case by comparing the GC peak areas of different solutions
containing the same concentration of a number of mono-oxygenated
terpenes including the alcohols, epoxides, and ketones. With this small
caveat in mind, mixtures containing different concentrations of (+)-
perillyl alcohol and all components of a reaction except NADH and
the substrate were extracted and analyzed as described above. The ratio
of the peak areas of (+)-perillyl alcohol and the internal standard was
plotted against the (+)-perillyl alcohol concentration. This calibration
plot was a straight line passing through the origin. The concentrations
of the different products formed, and hence the total product formation
rate, were readily determined from the plot. The coupling efficiency,
defined as the proportion of NADH consumed that lead to product
formation, was then readily calculated from the total product concentra-
tion and the amount of NADH added to initiate the reaction.
Characterization of Metabolites. The major (+)-R-pinene oxidation
products were initially identified by GC coelution experiments with
authentic samples. (+)-cis-Verbenol coeluted with (-)-cis-verbenol on
the DB-1 column but not the â-DEX column, which partially identified
the product. (+)-Myrtenol, (+)-verbenone, and the (+)-R-pinene
epoxides were identified by coelution on both columns. To establish
the identities of these compounds, a preparative scale oxidation reaction
was carried out with the Y96F/ V247L mutant.37 The products were
separated by silica column chromatography, eluting with hexane/ethyl
acetate mixtures. (+)-cis-Verbenol, (+)-verbenone, and (+)-myrtenol
were all readily separated and purified. Their 500-MHz 1H NMR spectra
were in full agreement with those of authentic compounds. The
diastereomers of the epoxide could not be separated but these had been
positively identified by coelution with a racemic commercial sample.
The other minor products could not be purified satisfactorily and were
not characterized further.
Figure 2. Active site structure of the monoclinic form of wild-type P450cam
with camphor bound, crystallized from PEG8000.27 The T101 side-chain
orientation is the same as that observed in the present work but rotated by
ca. 90° compared to the orthorhombic form.20
that (+)-R-pinene would also be bound with its C8,C9 methyl
groups contacting V295, and the overall orientation would be
approximately the same as that for camphor. The camphor
carbonyl group forms a hydrogen bond with Y96 and it also
contacts the F87 side chain.20 Since this carbonyl group is absent
from (+)-R-pinene, the Y96F mutation should improve pinene
binding by removing the polar phenol side chain.38 The larger
side chain of the F87W mutation might improve the enzyme-
substrate fit by “filling in” the space left as a result of the
absence of the camphor carbonyl group in (+)-R-pinene. The
effect of side-chain volume at the 87 position was also
investigated with the F87A and F87L mutations. The C10 methyl
group of camphor contacts the V247 side chain in the wild
type.20 Since the C10 methyl group of (+)-R-pinene is in a
different position in the molecule, the V247L mutation could
improve enzyme-substrate complementarity.
The mutants Y96F, F87A/Y96F, F87L/Y96F, F87W/Y96F,
Y96F/V247L, and F87W/Y96F/V247L were examined for the
binding and oxidation of (+)-R-pinene. All these mutants were
readily expressed and purified to homogeneity and showed
similar stability to the wild-type enzyme. A preliminary account
of some of this work has been published.39
(+)-r-Pinene Binding and Oxidation. The substrate binding
and catalytic parameters for (+)-R-pinene oxidation by wild-
type P450cam and the active site mutants are collected in Table
2. For comparison the camphor binding constant (KD) for wild-
type P450cam is 0.25 µM. The camphor oxidation rate of the
wild-type enzyme is 400 min-1 under identical conditions, with
>95% coupling, i.e., nearly all the NADH consumed is utilized
for the formation of 5-exo-hydroxycamphor.40 Wild-type P450cam
and all the mutants showed >70% conversion to high-spin heme
upon (+)-R-pinene binding. The structural similarity between
Results and Discussion
Designing the Active Site Mutations. As shown in Figure
1, (+)-R-pinene is structurally related to camphorscamphor is
a bicyclic-2,2,1 compound while (+)-R-pinene has the 3,1,1
structure. The carbonyl group in camphor is absent from (+)-
R-pinene and the C10 methyl group is on a different ring carbon
in (+)-R-pinene. We therefore set out to engineer the P450cam
active site to compensate for these structural differences so as
to promote (+)-R-pinene binding and oxidation.
In the crystal structure of wild-type P450cam with bound
camphor (Figure 2), the C8,C9 methyl groups of the substrate
are in van der Waals contact with the V295 side chain.
Camphane was bound in a virtually identical orientation.22 Our
starting point for active-site engineering was the assumption
(38) Atkins, W. M.; Sligar, S. G. J. Biol. Chem. 1988, 263, 18 842-18 849.
(39) Bell, S. G.; Sowden, R. J.; Wong, L.-L. Chem. Commun. 2001, 635-636.
(40) Gelb, M. H.; Heimbrook, D. C.; Ma¨lko¨nen, P.; Sligar, S. G. Biochemistry
1982, 21, 370-377.
(37) Harford-Cross, C. F.; Carmichael, A. B.; Allan, F. K.; England, P. A.;
Rouch, D. A.; Wong, L.-L. Protein Eng. 2000, 13, 121-128.
9
708 J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003

Molecular Recognition in (+)-R-Pinene Oxidation
A R T I C L E S
Table 2. Binding and Oxidation of (+)-R-Pinene by Wild-Type (WT) P450Cam and Active-Site Mutants
WT
Y96F F87A/Y96F F87L/Y96F F87W/Y96F Y96F/V247L F87W/Y96F/V247L F87W/Y96F/L244A/V247L F87W/Y96F/L244A Y96F/L244A/V247L
% high spin heme
KD, µM
85
1.10
82
95 70 80 95 95 75 85 65 90
0.15 0.12 0.10 0.08 0.14 0.30 0.24 0.26 0.15
NADH turnover ratea
147
56
210
150
163
88
171
96
298
271
129
65
233
156
122
120
223
129
product formation rateb 18.6
coupling efficiencyc
23%
38%
72%
54%
56%
91%
51%
67%
95%
58%
a Given as nanomoles of NADH consumed per nanomole of P450cam per minute. b The total amount in nanomole of product formed per nanomole of
P450cam per minute. c The coupling efficiency is the ratio of the total amount of products formed to the amount of NADH consumed, i.e., the yield based
on NADH, and is expressed as a percentage.
Table 3. Distribution of Products ((2%) from (+)-R-Pinene Oxidation Catalyzed by Wild-Type and Active Site Mutants of P450cam
P450cam enzyme
(+)-R-pinene epoxides
(+)-cis-verbenol
(+)-myrtenol
(+)-verbenone
others
wild-type
Y96F
F87A/Y96F
F87L/Y96F
F87W/Y96F
Y96F/V247L
F87W/Y96F/V247L
F87W/Y96F/L244A/V247L
F87W/Y96F/L244A
Y96F/L244A/V247L
4%
3%
22%
12%
7%
12%
3%
12%
2%
31%
46%
33%
38%
66%
55%
70%
44%
86%
55%
4%
10%
7%
7%
3%
10%
12%
8%
10%
7%
7%
8%
13%
5%
32%
51%
29%
30%
33%
17%
19%
15%
31%
5%
7%
4%
2%
1%
12%
Scheme 1. Products from (+)-R-Pinene Oxidation Catalyzed by
Cytochrome P450cam
camphor and (+)-R-pinene also meant that wild-type P450cam
showed tighter (+)-R-pinene binding, higher oxidation activity,
and tighter coupling than for compounds such as styrene,41-43
ethylbenzene,44 and linear alkanes,36 which have very different
structures from camphor.
As predicted the Y96F mutation strengthened (+)-R-pinene
binding and increased the rate and coupling of oxidation. A
hydrophobic substitution at F87, regardless of side-chain
volume, further enhanced both the binding and oxidation
activity. On the other hand, adding the V247L mutation did
not significantly tighten (+)-R-pinene binding but both the rate
and coupling were greatly improved. The (+)-R-pinene oxida-
tion rate of 270 min-1 for the Y96F/V247L double mutant was
ca. 70% of the camphor oxidation activity of wild-type P450cam
under identical conditions.
The data showed that there is no direct correlation between
the high-spin heme content and the strength of (+)-r-pinene
binding or the rate and coupling efficiency. It is also interesting
to note that (+)-R-pinene binding by Y96F and a number of
multiple mutants was tighter than camphor binding by the wild-
type.
(+)-r-Pinene Oxidation Products. Camphor oxidation by
wild-type P450cam occurs exclusively at C5 to give the exo
alcohol. If the geminal methyl groups of camphor and (+)-R-
pinene were superimposed in the crystal structure, the dominant
product should be (+)-cis-verbenol (Scheme 1). In addition
some (+)-R-pinene epoxide might be expected because the
reactive olefinic double bond might also be close to the heme
iron.
GC coelution experiments and 1H NMR spectroscopy showed
that the major product from (+)-R-pinene oxidation catalyzed
by wild-type P450cam and all the mutants was indeed (+)-cis-
verbenol. However, despite the similar structures of camphor
and (+)-R-pinene, the selectivity for (+)-cis-verbenol was
nowhere near the 100% selectivity for camphor oxidation. Both
the cis and trans isomers of (+)-R-pinene epoxide were minor
products, identified by coelution with an authentic sample. This
is an important observation because it indicated that the (+)-
R-pinene substrate was not freely mobile within the active site.
If the substrate was freely mobile, the most reactive functional
groupsthe olefinic double bondswould be preferentially at-
tacked and the epoxides would be the major products. (+)-
Myrtenol, which arose from oxidation of the C10 methyl group,
was another minor product identified by GC coelution experi-
1
ments and its H NMR spectrum. (+)-Verbenone, the further
oxidation product of (+)-cis-verbenol, was also formed. In
addition to these characterized compounds there were additional,
small peaks in the gas chromatograms. These products were
not characterized but some peaks were at retention times
consistent with monooxygenated (+)-R-pinene compounds,
while the longer retention times of others were consistent with
compounds derived from oxidation at multiple carbons.
The distribution of (+)-R-pinene oxidation products for the
P450cam enzymes is given in Table 3. Wild-type P450cam showed
some selectivity for (+)-cis-verbenol, but there were many other
products, especially compounds at long retention times. The
Y96F mutation increased the proportion of (+)-cis-verbenol and
(+)-myrtenol, mainly at the expense of the multiple oxidation
products. The F87A and F87L mutations, which were expected
to increase the space available in the active site and potentially
increase substrate mobility, increased the amount of epoxides
formed. In contrast, the F87W and V247L mutations, which
reduced the active site volume, significantly increased the
selectivity for (+)-cis-verbenol. The effects of these two
(41) Nickerson, D. P.; Harford-Cross, C. F.; Fulcher, S. R.; Wong, L. L. FEBS
Lett. 1997, 405, 153-156.
(42) Fruetel, J. A.; Collins, J. R.; Camper, D. L.; Loew, G. H.; Ortiz de
Montellano, P. R. J. Am. Chem. Soc. 1992, 114, 6987-6993.
(43) Mayhew, M. P.; Reipa, V.; Holden, M. J.; Vilker, V. L. Biotechnol. Prog.
2000, 16, 610-616.
(44) Filipovic, D.; Paulsen, M. D.; Loida, P. J.; Sligar, S. G.; Ornstein, R. L.
Biochem. Biophys. Res. Commun. 1992, 189, 488-495.
9
J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003 709

A R T I C L E S
Bell et al.
Figure 3. Stereoview of the active site structure of molecule A of the F87W/Y96F/V247L mutant of P450cam showing the substrate electron density and
the fit to the (+)-R-pinene substrate. The (+)-R-pinene location overlaps that of camphor in the wild-type structure and the (+)-R-pinene orientation can be
generated approximately from that of camphor by a number of rotations. See text for details. The figure was generated by MOLSCRIPT and BOBSCRIPT
and rendered by RASTER3D.
Table 4. Contacts (4 Å or Less) between (+)-R-Pinene and the
mutations were not additiveswhen they were combined the
selectivity for (+)-cis-verbenol was only increased slightly.
Nevertheless the selectivity for (+)-R-pinene oxidation at C4,
i.e., forming verbenol and verbenone, was greatly improved
compared to the that for wild-type enzyme, to 78% and 72%
respectively for the F87W/Y96F/V247L and F87W/Y96F
mutants.
F87W/Y96F/V247L Mutant of P450cam in Molecules A and B
molecule A
P450cam
molecule B
P450cam
(+)-R-pinene
distance
distance
C-1
CH2 Trp87
CD2 Leu247
4.00
3.90
C1A heme
NA heme
C4A heme
CA Gly248
ND heme
NA heme
CA Gly248
N Gly248
3.98
3.71
3.90
3.90
3.70
3.75
3.90
3.69
3.55
3.55
3.56
C-2
Crystal Structure of the F87W/Y96F/V247L Mutant with
(+)-r-Pinene Bound. We determined the 2.3-Å resolution
crystal structure of the F87W/Y96F/V247L mutant with (+)-
R-pinene bound in its active site. This triple mutant was
marginally the most selective of the P450cam enzymes initially
studied. The structure should provide insight into the enzyme-
substrate contacts and how product selectivity could be further
engineered. Crystals of the mutant were obtained by crystal-
lization with PEG8000. The mutant crystallized in the space
group P21, identical to the monoclinic form obtained from
PEG4000 and with virtually identical unit cell parameters27 but
different from the orthorhombic crystals from ammonium
sulfate.20
The structure was solved by molecular replacement based
on the phases from the wild-type structure. The two independent
molecules (A and B) in the unit cell were readily located and
refined. Most of the CR backbone and side chains of the two
molecules were superimposable on that of the wild-type. The
iron was below the plane of the heme (0.36 Å in both molecules
A and B, 0.37 Å in the wild-type) and the iron-cysteine thiolate
distances (2.08 Å in A and 2.29 Å in B) were similar to that in
the wild-type (2.20 Å). The key heme-protein contacts found
in the wild-type structure were all present.
There were no water molecules in the active site of either
mutant molecule in the unit cell. Most of the side-chains in the
active site were found in similar or identical orientations to the
wild-type, suggesting that the three mutations had little effect
on the structure. The (+)-R-pinene substrate was bound in
different orientations in the two molecules in the unit cell, but
its location in both molecules overlapped significantly with that
found for camphor in the wild-type structure.
(+)-r-Pinene Binding in Molecule A. The (+)-R-pinene
binding orientation in molecule A (Figure 3) was closely related
to that found for camphor and camphane in the wild-type
structures.22 Camphor and camphane are located over the meso
carbon CHA and the adjacent C1A and C4D of the A and D
C-3
C-4
C-5
CG2 Thr252
3.20
NA heme
ND heme
NA heme
ND heme
CHA heme
C1A heme
C4D heme
3.51
3.65
3.70
3.53
3.72
3.73
3.61
CD1 Leu244
CD2 Leu247
CH2 Trp87
C-6
C1A heme
C2A heme
3.98
4.00
C-7
C-8
CD1 Leu244
CG1 Val295
C1A heme
C2A heme
C3A heme
CBA heme
3.65
3.82
3.97
3.77
3.89
3.78
CG2 Thr101
C1A heme
C2A heme
C4D heme
CBA heme
CHB heme
CG1 Val295
OD2 Asp297
CBA heme
C1A heme
C4A heme
C1B heme
NA heme
3.98
3.34
3.48
3.98
3.00
3.30
3.59
3.61
3.61
3.99
3.92
3.96
3.21
3.06
3.00
3.06
C-9
CG2 Thr101
3.64
C-10
CG2 Thr185
CD2 Leu247
CG Leu247
CG2 Val396
3.71
3.72
3.67
3.36
NB heme
NC heme
ND heme
pyrroles,20 with the C8,C9 methyls above the A pyrrole and in
contact with V295. The C1 atom of (+)-R-pinene was at almost
the same location as C1 of camphor in the wild-type structure.
The (+)-R-pinene C10 methyl was in van der Waals contact
(Table 4) with the side chains of T185, L247, and V396, which
were located high up in the substrate pocket. The indole side
chain of W87 contacted the (+)-R-pinene C1, suggesting that
this larger side chain compensated for the absence of the
camphor carbonyl group. Closer to the heme, the L244 side
chain, which played a very important role in camphor binding
9
710 J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003

Molecular Recognition in (+)-R-Pinene Oxidation
A R T I C L E S
Figure 4. Comparison of the (+)-R-pinene binding location and orientation in molecule A (panels a and b) and molecule B (panels c and d) of the F87W/
Y96F/V247L mutant with that of camphor in the wild-type structure. Panels a and c are views from the top of the active site to emphasize the contacts
between (+)-R-pinene and side-chains close to the heme. Panels b and d are views from the side to show the location of the T252 side chain and emphasize
contacts with side chains near the top of the active site. The figures were generated by the program WEBLAB VIEWER PRO.
in the wild-type, only contacted the C7 carbon of (+)-R-pinene
in the mutant.
shown in Figure 5. This unexpected orientation was related to
that in molecule A by a ca. 180° rotation about the vector
bisecting the C8,C6,C9 plane (Figure 4). The C1 and C10 atoms
moved from the top of the active site to positions close to the
heme. The C8 and C9 methyls effectively switched positions
compared to molecule A, such that C8 in molecule B contacted
T101, while C9 contacted V295 (Table 4). In addition, the (+)-
R-pinene molecule moved toward pyrrole A such that C9 was
close to one of the carboxylate oxygens of D297, and both C8
and C9 were in contact with CBA of the propionate group on
pyrrole A.
The (+)-R-pinene binding orientation in molecule A could
be generated from that of camphor in the wild-type structure
by aligning the pinene C1,C5 vector with the C1,C4 vector of
camphor and rotating the (+)-R-pinene molecule around this
vector by ca. 90°, so that the C8,C9 methyl groups moved away
from V295 and toward the CHA meso carbon (Figure 4). As a
result the C8 methyl group in (+)-R-pinene contacted the V295
side chain, but the C9 methyl contacted T101 (Table 4). The
rotation was also accompanied by a tilt of the pinene C1,C5
vector, with C5 moving toward the D pyrrole compared to the
position of camphor C4 in the wild-type structure. The combined
rotation and tilting placed the C4 atom of (+)-R-pinene closest
to the heme iron. The Fe-C4 distance was 3.40 Å and the C/Fe/
S(C357) angle was 175°, compared with 3.40 Å and 164° for
camphor C5 in the wild-type structure.20 The exo face at C4
was closer to the iron than the endo side. The predicted product
would therefore be (+)-cis-verbenol. We note that, if no tilting
of (+)-R-pinene relative to camphor occurred, the rotation on
its own would have brought the exo side at C4 past the iron.
The endo side would then be closer to the oxygen of the ferryl
intermediate, and (+)-trans-verbenol would have been the major
product.
The carbon atom closest to the iron was the C10 methyl (2.70
Å, C/Fe/S angle 170°), and (+)-myrtenol should be the major
product.
Enzyme-Substrate Contacts and Implications in Cou-
pling and Product Selectivity. The initial assumption in
designing P450cam active-site mutations was that the structural
resemblance to camphor should allow (+)-R-pinene to bind in
a similar orientation to that found for camphor in the wild-type.
This was largely the case in molecule A but not in molecule B.
Indeed the (+)-R-pinene/enzyme contacts in both molecules
were significantly different from those in the wild-type structure.
Due to the rotation of (+)-R-pinene in molecule A relative to
the camphor binding orientation in the wild-type, only one,
rather than both, of the C8,C9 methyl groups contacted V295.
The side-chains at the 87, 96, and 244 positions, which were
(+)-r-Pinene Binding in Molecule B. The electron density
map and fit for the (+)-R-pinene substrate in molecule B is
9
J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003 711

A R T I C L E S
Bell et al.
Figure 5. Stereoview of the active-site structure of molecule B of the F87W/Y96F/V247L mutant of P450cam showing the substrate electron density and the
fit to the (+)-R-pinene substrate. The (+)-R-pinene location overlaps that found in molecule A and of camphor in the wild-type structure. The (+)-R-pinene
orientation can be generated approximately from that in molecule A by rotation. See text for details. The figure was generated by MOLSCRIPT and BOBSCRIPT
and rendered by RASTER3D.
prominent in camphor binding by the wild-type,20 were much
less involved in (+)-R-pinene binding by the mutant. In contrast
the T101 side-chain was not involved in camphor binding in
the wild-type, but in the mutant it contacted one of the (+)-R-
pinene geminal methyl groups. There were also more (+)-R-
pinene/heme contacts in both molecules than between camphor
and the heme, in particular in molecule B (Table 4).
The absence of water molecules in the active site of the
mutant with (+)-R-pinene bound was consistent with the large
shift of the heme spin state to high spin and the relatively high
coupling efficiency of 51%. However, since there was no
evidence for the binding of water to the heme iron in either
molecule, the crystal structure did not offer an explanation to
why the heme was not fully high spin. A possible explanation
is that the structure was determined at -100 °C, while the heme
spin state was measured at 30 °C. At this higher temperature it
is very likely that the (+)-R-pinene molecule undergoes dynamic
motions and, in some binding orientations, water may bind to
the iron to give a population of low-spin heme.
two orientations would be a ca. 180° rotation about the vector
bisecting the C8,C6,C9 plane (Figure 4). During this process the
C10 methyl would traverse the space between the heme iron
and the T252 side chain, which had been proposed to be the
oxygen binding pocket,21 and uncoupling was expected.
Identification of substrate binding orientations allowed pre-
dictions of the (+)-R-pinene oxidation products. We would
predict that (+)-cis-verbenol should be the major product from
molecule A, and for molecule B (+)-myrtenol should be the
major product. Experimentally we found that the proportion of
(+)-cis-verbenol (70%) far exceeded that of (+)-myrtenol (4%).
Since interconversion between the two binding orientations was
expected at the assay temperature, this result might simply reflect
the higher reactivity of the sp2 C-H bonds at C4 compared to
the sp3 bonds at C10. Alternatively, the expected uncoupling
for the binding orientation in molecule B might also reduce the
amount of (+)-myrtenol formed. In addition, we cannot rule
out the possibility that the binding orientation in molecule A
was slightly more stable and therefore more prevalent in solution
at the assay temperature.
Dynamic substrate binding and the entry of water to bind to
the heme iron may partially explain why the coupling for (+)-
R-pinene oxidation by the F87W/Y97F/ V247L mutant was
lower than might be expected from the crystal structure. Thus
access of water to the heme iron during the catalytic cycle could
lead to protonation of the ferric peroxide intermediate at the
heme-bound oxygen and subsequent loss of hydrogen per-
oxide.45 In addition, Sligar and Poulos have both suggested that
substrate mobility, regardless of whether water was bound to
the heme iron in some population of the enzyme, may also lead
to uncoupling.22,45 This mechanism was expected to become
more important if some of the interconverting binding orienta-
tions, or movement between them, involved close approach of
the substrate to the heme-bound oxygen species. Of the two
observed (+)-R-pinene binding orientations, that in molecule
A resembled that of camphor in the wild-type and little
uncoupling was expected. On the other hand the close approach
of the C10 methyl group to the heme iron in molecule B could
interfere with the iron-oxygen interaction, leading to uncou-
pling. Furthermore the crystal structure suggested that the most
direct and likely pathway for the interconversion between the
Another factor is the known movement of camphor during
the catalytic cycle. Camphor moves toward the heme meso
carbon CHA in the wild-type structure to make room for oxygen
to bind to the ferrous heme.27 After O-O bond activation and
formation of the ferryl intermediate, camphor moves back
toward the iron.27 During this process the exo face at C5 is
always closer to the iron, and 5-exo-hydroxycamphor is the only
product. In an analogous manner we would expect the binding
orientation in molecule A to be maintained and give (+)-cis-
verbenol as the product. The situation for molecule B is more
complicated because the C10 methyl was very close to the iron
and had to move over a longer distance upon oxygen binding.
The interesting question is whether this movement resulted in
other orientations being adopted; e.g., a certain population of
pinene molecules in molecule B might adopt the orientation
found in molecule A, thus leading to more (+)-cis-verbenol. It
is also easy to envisage orientations which would bring the
olefinic double bond close to the iron, resulting in epoxide
formation. We believe that a combination of the inherent
reactivity of the pinene C-H bonds and substrate movement
during the catalytic cycle probably accounted for the observed
product distribution.
(45) Loida, P. J.; Sligar, S. G. Biochemistry 1993, 32, 11 530-11 538.
9
712 J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003

Molecular Recognition in (+)-R-Pinene Oxidation
A R T I C L E S
Further Engineering of P450cam. Based on the crystal
structure of the F87W/Y96F/V247L mutant and the selectivity
of the other mutants, we sought combinations of mutations
which might further increase the selectivity for (+)-cis-verbenol
formation. The selectivity data clearly showed that the Y96F,
F87W, and V247L mutations all promoted the formation of (+)-
cis-verbenol, with the F87W mutation apparently having the
greater effect. However, the effects of the F87W and V247L
mutations were not additive. The crystal structure suggested a
plausible explanation: the leucine side-chain at the 247 position
contacted the C10 methyl and pulled the (+)-R-pinene molecule
away from the side-chain at the 87 position. In other words
there was competition rather than cooperation between the
effects of the F87W and V247L mutations.
camphor oxidation by the wild-type and the Y96F mutant38 or
any of the mutants studied here; 5-oxocamphor was not formed
until >95% of the camphor had been consumed. Further
oxidation would occur if binding of the initial alcohol product
could compete with binding of the starting substrate. Camphor
binding by wild-type P450cam is more than an order of magnitude
tighter than the binding of 5-exo-hydroxycamphor.29 It has been
reported that in vivo camphor oxidation in E. coli by a triple
fusion protein, in which P450cam was fused with its two
physiological electron-transfer partner proteins, gave 5-oxo-
camphor before camphor was exhausted.46 The change from in
vitro to in vivo conditions could have resulted in the increased
double oxidation of camphor. However, another in vivo P450cam
system, in which the three proteins were separately expressed
and thus freely diffusing inside the E. coli cytoplasm, did not
give the ketone before camphor exhaustion.47 These data
indicated that there was a smaller difference between the strength
of camphor and 5-exo-hydroxycamphor binding by the P450cam
domain in the triple fusion protein, perhaps arising from
structural changes induced by fusing P450cam with the electron-
transfer proteins in a single polypeptide. It is not clear why the
combination of mutations in the Y96F/L244A/V247L mutant
also narrowed this difference in binding so that this mutant was
more effective in double oxidation of (+)-R-pinene to (+)-
verbenone than the Y96F/V247L and F87W/Y96F/L244A
mutants. Unraveling the intricate enzyme/substrate interactions
and associated dynamics that give rise to this complex activity
pattern will require further investigations, including X-ray
crystallography. However, the results suggest that it should be
possible to engineer a P450cam mutant that will stop the oxidation
at (+)-cis-verbenol, while another mutant could oxidize (+)-
R-pinene to (+)-verbenone even in the presence of excess
pinene.
We sought to increase the selectivity for (+)-cis-verbenol
formation by disfavoring the binding orientation in molecule
B. The enzyme/substrate contacts from the crystal structure
suggested that this might be possible if the (+)-R-pinene
molecule was to move toward pyrrole D, thus weakening or
breaking the contacts with the D297 side-chain and the
propionate CBA carbon. The L244 side chain was targeted
because it was located over pyrrole D and it also contacted the
(+)-R-pinene molecule. We therefore introduced the L244A
mutation and prepared the F87W/Y96F/L244A/V247L, F87W/
Y96F/L244A, and Y96F/L244A/V247L mutants, which also
allowed us to explore the possible competition between the
effects of the F87W and V247L mutations. The (+)-R-pinene
oxidation activity and selectivity of these new mutants are given
in Tables 2 and 3.
Addition of the L244A mutation increased the activity of the
F87W/Y96F and F87W/Y96F/V247L mutants but not the Y96F/
V247L mutant. When the V247L mutation was present, the
L244A mutation had little effect on the strength of pinene
binding. On the other hand the F87W/Y96F/L244A mutant
showed much weaker pinene binding than the F87W/Y96F
parent mutant. Interestingly, the tight coupling of the F87W/
Y96F/L244A mutant did not require the complete shift of the
heme spin state to high spin. This result mirrored that of
camphor oxidation by the Y96F mutant, where complete
coupling was observed for a 55% high-spin heme content in
the camphor-bound form.38 The L244A mutation also had
significant effects on the selectivity, notably reducing the amount
of (+)-myrtenol formed. Moreover, the F87W/Y96F/L244A
mutant gave 86% (+)-cis-verbenol, the highest selectivity of
all the P450cam mutants studied. With 5% of (+)-verbenone also
formed, the overall regioselectivity for C4 attack was 91%. This
high selectivity and tight coupling (>95%) were close to those
for camphor oxidation by the wild-type, although the product
formation rate was lower. The Y96F/L244A/V247L mutant gave
55% (+)-cis-verbenol and 32% (+)-verbenone, and there was
one major side product (ca. 10%) at a long retention time. The
data for the new mutants suggested that the binding orientation
in molecule B was disfavored by the L244A mutation but not
when both the F87W and V247L mutations were present, again
reflecting the competition between the effects of these two
mutations.
Conclusions
The heme monooxygenase cytochrome P450cam has been re-
engineered for the oxidation of (+)-R-pinene which is structur-
ally related to camphor, the natural substrate of the enzyme.
Consideration of the structures of these two molecules lead to
mutants with enhanced activity and product selectivity for (+)-
R-pinene oxidation. The crystal structure of the Y96F/F87W/
V247L mutant provided new insight into P450cam substrate
binding and catalysis. Analysis of the enzyme-substrate
interactions and dynamics lead to the introduction of the L244A
mutation, which increased the selectivity for (+)-cis-verbenol
to 86% for the F87W/ Y96F/L244A mutant, but unexpectedly
there was significant further oxidation by the Y96F/L244A/
V247L mutant, which gave 32% (+)-verbenone. The P450cam
system has also been expressed in E. coli to provide whole-
cell, in vivo substrate oxidation systems.46,47 With some
improvements in selectivity, engineered P450cam mutants may
have applications in the biotransformation of (+)-R-pinene.
Finally the results of the present work also suggest that the
current understanding of P450 catalytic activity allows the design
of mutations to increase the rate of oxidation of many unnatural
substrates on the basis of the hydrophobicity, size, shape, and
rigidity of the target compound. However, the much more
We found that (+)-cis-verbenol was further oxidized to (+)-
verbenone to various extents by all the P450cam enzymes studied,
especially the Y96F/L244A/V247L mutant, even though (+)-
R-pinene was present in excess. This was not the case for
(46) Sibbesen, O.; De Voss, J. J.; Ortiz de Montellano, P. R. J. Biol. Chem.
1996, 271, 22 462-22 469.
(47) Bell, S. G.; Harford-Cross, C. F.; Wong, L. L. Protein Eng. 2001, 14, 797-
802.
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A R T I C L E S
Bell et al.
demanding challenge is in engineering the regioselectivity and
(to Z.R.): Project “973” No. G1999075602 and Project “863”
No. 2001AA233011. We thank the support of the Biotechnology
and Biological Sciences Research Council, U.K. (Grant Refer-
ence B10666), and the Engineering and Physical Sciences
Research Council, U.K. (Studentship to R.J.S.). We also thank
the Referees for helpful suggestions.
stereoselectivity of substrate oxidation because the P450cam
/
substrate molecular recognition interactions are both highly
dynamic and subtly modulated by amino acid substitutions.
Acknowledgment. This research was supported by the
following grants from the National Science Foundation of China
JA028460A
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714 J. AM. CHEM. SOC. VOL. 125, NO. 3, 2003