Scheme 1
of (1). The monocyclic compound (2) could also be bound in a
similar orientation to camphor if it adopted the slightly higher
energy conformation shown in Fig.1, and these mutants may
also have higher activities for the oxidation of (2).
The substrate binding and catalytic parameters for the
oxidation of (1) and (2) by wild-type P450cam and the mutants
are given in Table 1. The monocyclic (2) was a very poor
proportion (16%) of (9), while the less active wild-type and
Y96F mutant gave the most epoxide (17 and 26% respectively).
The predicted major product from the proposed binding
orientation was (2)-cis-isopiperitenol, and some (S)-limonene
epoxide was also expected. The observation of the trans isomer
(7) and also some carveol product suggested that the more
conformationally mobile (2) did not adopt the camphor binding
orientation, and that there were multiple substrate binding
modes. We note that the P450 enzyme from peppermint
oxidises (2) with total selectivity to give the trans-isopiperitenol
(7), while the spearmint enzyme gives only trans-carveol
substrate for the wild-type compared to camphor (K
D
=
2
1
0.25mM, rate = 400 min under identical conditions, 100%
coupling efficiency). However, the bicyclic (1) was bound
much more tightly and oxidised at a faster rate with higher
coupling than (2), in all likelihood reflecting the closer structure
of (1) to camphor. As predicted the Y96F mutation strengthened
the binding and also increased the rate and coupling for the
oxidation of both substrates, particularly for (2). The addition of
the F87W or V247L mutation further enhanced both the binding
and oxidation activity for (1), suggesting improved enzyme–
substrate fits in the F87W–Y96F and Y96F–V247L double
mutants. Interestingly (1) was bound more tightly by the Y96F
and these two double mutants than camphor was by the wild-
11,12
(9).
In summary the results suggest that the strategy of designing
mutations based on the structure of the monoterpenes and
potential side-chain/substrate contacts to improve the enzyme–
substrate fit was very successful in promoting monoterpene
oxidation by P450cam. In addition, with some further selectivity
engineering, P450cam variants may have applications in the
biotransformation of terpenes in fine chemical synthesis.
Finally, since not all of the limonene and pinene oxidation
products have been utilised by nature, the oxidation of these and
indeed other terpenes by engineered P450 enzymes could give
rise to novel fragrances and flavourings or new biologically
active compounds.
We thank the Higher Education Funding Council for England
for support of this work, and the referees for helpful comments.
SGB thanks the Biotechnology and Biological Sciences Re-
search Council, UK, and RJS the Engineering and Physical
Sciences Research Council, UK, for studentships.
2
1
type, and the fast substrate oxidation rate of 271min and near
total coupling efficiency of the Y96F–V247L double mutant
almost matched the camphor oxidation activity of wild-type
P450cam. For (2) the addition of the F87W and V247L mutations
slightly weakened substrate binding but substantially increased
the rate and coupling, and so although (2) was less tightly bound
it was located closer to the haem resulting in more efficient
substrate oxidation. The F87W–Y96F–V247L triple mutant
showed weaker monoterpene binding and lower activity
compared to the double mutants, probably due to steric
hindrance between (1) and (2) and the much smaller active site
cavity in this mutant.
Notes and references
5
Since camphor is selectively oxidised at C to give the exo
alcohol, the major products from the proposed binding
orientation of (1) should be (+)-cis-verbenol (3) and a-pinene
epoxide (4) (Fig.1, Scheme1). GC co-elution experiments
showed that (3) was indeed the major product ( > 60%) for all
the P450cam enzymes. The cis and trans isomers of (4) were
minor products (total < 8%), and the enzymes showed little
selectivity between the two. In addition (+)-myrtenol (5), which
arose from oxidation of the allylic methyl group, and verbenone
1
J. Gershenzon and R. B. Croteau, Lipid Metab. Plants, 1993, 339.
2 S. Arctander, Perfume and Flavor Materials of Natural Origin, Allured,
Wheaton IL, 1960.
3 B. V. Charlwood and K. A. Charlwood, Monoterpenoids, 1991, 7, 43;
B. M. Fraga, Sesquiterpenoids, 1991, 7, 45.
4
F. Karp, C. A. Mihaliak, J. L. Harris and R. B. Croteau, Arch. Biochem.
Biophys., 1990, 276, 219.
C. Haudenschild, M. Schalk, F. Karp and R. B. Croteau, Arch. Biochem.
Biophys., 2000, 379, 127.
I. C. Gunsalus and G. C. Wagner, Methods Enzymol., 1978, 52, 166.
S. G. Sliga and R. I. Murray, in Cytochrome P450: Structure,
Mechanism and Biochemistry, ed. P. R. Ortiz de Montellano, Plenum,
New York, 1st edn., 1986.
5
(6), the further oxidation product of (3), were also formed
(Scheme 1). The most active mutant Y96F–V247L gave 70%
(3) and 7% each of (5) and (6), while the less active triple mutant
6
7
F87W–Y96F–V247L was the most selective, giving 85% (3).
The selectivity for the oxidation of (2) was more sensitive to
the mutations. Chiral-phase GC analysis showed that the main
products were (2)-trans-isopiperitenol (7), (2)-cis-limonene
epoxide (8), and (2)-trans-carveol (9) (Scheme1). All the
P450cam enzymes had very high diastereoselectivity ( > 95% by
GC) for the formation of all these products. Mutants containing
the F87W mutation were more selective for (7) (82% for both
the F87W–Y96F and F87W–Y96F–V247L mutants). The most
active mutant Y96F–V247L gave 70% (7) but also the highest
8 E. J. Mueller, P. J. Loida and S. G. Sligar, in Cytochrome P450:
Structure, Mechanism and Biochemistry, ed. P. R. Ortiz de Montellano,
Plenum, New York, 2nd edn., 1995.
9
T. L. Poulos, B. C. Finzel and A. J. Howard, J. Mol. Biol., 1987, 195,
87.
6
1
1
0 W. M. Atkins and S. G. Sligar, J. Biol. Chem., 1988, 263, 18842.
1 R. B. Croteau and J. Gershenzon, Recent Adv. Phytochem., 1994, 28,
1
93.
12 M. Schalk and R. B. Croteau, Proc. Natl. Acad. Sci. USA, 2000, 97,
11948.
636
Chem. Commun., 2001, 635–636
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