872 J. Am. Chem. Soc., Vol. 120, No. 5, 1998
Buist and Behrouzian
Scheme 1
could be to act as an electron sink,24 or it could be more
intimately involved in the hydrocarbon activation step.18
On the basis of Scheme 1, one would predict that if a
desaturase could be induced to act as an oxygenase, the
regioselectivity of oxygenation should match that of the
hydrogen abstraction step involved in the corresponding de-
saturation reaction. We have demonstrated that this is indeed
the case using an in vivo, S. cereVisiae ∆9 desaturase system:
9-thia fatty acid analogues such as 3 were consistently oxygen-
ated more efficiently than the corresponding 10-thia analogues
to give the corresponding sulfoxides with the correct absolute
stereochemistry and with very high enantiomeric purity.25 The
site of initial oxidative attack for the parent ∆9 desaturation
reaction was subsequently found to be C-9, as anticipated, using
a KIE approach.26 In this study, we took advantage of the fact
that according to our mechanistic scheme, initial hydrogen
abstraction should be slow relative to the second C-H bond
cleavage and, a priori, more sensitive to isotopic substitution.
Thus a large primary deuterium isotope effect was observed
for C-H bond cleavage at C-9 while a negligible isotope effect
was obtained for the C10-H bond breaking step.
type and the much better studied biohydroxylation of unactivated
hydrocarbons follow similar reaction mechanisms. A possible
mechanistic scheme16 relating hydroxylation and desaturation
is shown in Scheme 1: one might envisage an initial hydrogen
atom abstraction step by a hypervalent iron-oxo species which
generates a carbon-centered radical “intermediate”17 or its iron-
bound equivalent19 (not shown). This species could collapse
to olefin via a one electron oxidation/deprotonation sequence
(pathway a),20 simple disproportionation (pathway b),21 or by a
rapid Lewis acid (Fe3+)-catalyzed dehydration of an alcohol
intermediate (pathway c).22 The role of the second iron atom
Another example of an apparent diverted desaturation is
evident in the biosynthesis of ricinoleic acid (5, (R)-12-
hydroxyoleic acid)sa rare but important natural product which
accumulates in the seed oil of the castor plant (Ricinus communis
L.). Ricinoleic acid has been termed “one of the world’s most
(10) Shanklin, J.; Achim, C.; Schmidt, H.; Fox, B. G.; Mu¨nck, E. Proc.
Nat. Acad. Sci. USA 1997, 94, 2981.
(11) The evidence includes the following (a) the diiron active site of the
soluble plant ∆9 desaturase is similar to that found in methane monooxy-
genase (sMMO)12san enzyme which hydroxylates alkanes; (b) the highly
conserved multi-His motif of the membrane-bound deaturases is also found
in the alkane ω-hydroxylase from P. oleoVorans;10 (c) hepatic cytochrome
versatile natural products” 27a because it is used directly as a
component of lubricants, soaps, adhesives, inks, comestics, etc.,
and also serves as an industrial feedstock for a variety of
important materials such plasticizers, Nylon-11, polyesters, and
polyurethanes.27b Interest in the enzyme system responsible for
the synthesis of this hydroxy acid has intensified recently in
the context of efforts to broaden the scope of its agricultural
production.27a It has been shown that ricinoleic acid is produced
by a membrane-bound oleate 12-hydroxylase which bears a
striking resemblance to the more common oleate ∆12-desaturase
in terms of the stereochemistry of C-H cleavage,28a,b substrate
requirements,28c and molecular weight.28d On the basis of a
detailed comparison between the amino acid sequence of the
microsomal Arabidopsis ∆12 oleate desaturase and that of castor
oleate hydroxylase, van de Loo et al. have suggested that
ricinoleic acid is produced by a subtly modified oleate ∆12
desaturase.28d We thought it would be interesting to determine
whether the putative “parent” oleate ∆12 desaturase also initiates
the oxidation of substrate at C-12. In this article, we document
P
450sa known hydroxylator also dehydrogenates selected substrates;13 (d)
a mutated yeast ∆22 sterol desaturase yields a 23-hydroxy sterol;14 and (e)
clavaminic acid synthase can either act as a hydroxylase or a desaturase
depending on the nature of the substrate.15
(12) Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P.
Nature 1993, 366, 537.
(13) Ortiz de Montellano, P. R. Trends Pharmacol. Sci. 1989, 10, 354.
(14) Hata, S.; Nishino, T.; Oda, Y.; Katsuki, H.; Aoyama, Y.; Yoshida,
Y. Tetrahedron Lett. 1983, 24, 4729.
(15) Lawlor, E. J.; Elson, S. W.; Holland, S.; Cassels, R.; Hodgson, J.
E.; Lloyd, M.; Baldwin, J. E.; Schofield, C. J. Tetrahedron 1994, 50, 8737.
(16) We have depicted the oxidant as a diiron species since very recent
Mo¨ssbauer studies10 have shown that the closely related alkane ω-hydroxy-
lase possesses a catalytically active diiron site.
(17) (a) Groves, J. T.; McClusky, G. A.; White, R. E.; Coon, M. J.
Biochem. Biophys. Res. Commun. 1978, 81, 154. (b) Bowry, V. W.; Lusztyk,
J.; Ingold, K. U. J. Am. Chem. Soc. 1991, 113, 5699. (c) Newcomb has
suggested that this species is not a true intermediate but behaves as “a
component of a reacting ensemble” with a very short lifetime; accordingly
it has been postulated that this type of hydrocarbon activation proceeds by
“a nonsynchronous, concerted oxygen insertion” mechanism: cf., Newcomb,
M.; Le Tadic-Biadetti, F. H.; Chestney, D. L.; Roberts, E. S.; Hollenberg,
P. F. J. Am. Chem. Soc. 1995, 117, 12085. Additional support for such a
mechanism as it applies to sMMO has recently been obtained using chiral
methyl group experiments.18
(18) Valentine, A. M.; Wilkinson, B.; Liu, K. E.; Komar-Panicucci, S.;
Priestley, N. D.; Williams, P. G.; Morimoto, H.; Floss, H. G.; Lippard, S.
J. J. Am. Chem. Soc. 1997, 119, 1818.
(19) (a) Siegbahn, P. E. M.; Crabtree, R. H. J. Am. Chem. Soc. 1997,
119, 3103. (b) Blackburn, J. M.; Sutherland, J. D.; Baldwin, J. E.
Biochemistry 1995, 34, 7548. (c) Baldwin, J. E.; Adlington, R. M.;
Marquess, D. G.; Pitt, A. R.; Porter, M. J.; Russell, A. T. Tetrahedron 1996,
52, 2525.
(23) (a) Buist, P. H.; Behrouzian B.; Alexopoulos, K. A.; Dawson B.;
Black, B. J. Chem. Soc., Chem. Commun. 1996, 2671. (b) Light, R. J.;
Lennarz, W. J.; Bloch, K. J. Biol. Chem., 1962, 237, 1793.
(24) Que, L., Jr.; Dong, Y. Acc. Chem. Res 1996, 29, 190.
(25) (a) Buist, P. H.; Marecak, D. M. J. Am. Chem. Soc. 1992, 114,
5073. (b) Buist, P. H.; Marecak, D. M. Can. J. Chem. 1994, 72, 176. (c)
Buist, P. Marecak, D.; Dawson B.; Black, B. Can. J. Chem. 1996, 74, 453.
(26) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1996, 118, 6295.
(27) (a) Somerville C.; Browse J. Science 1991, 252, 80. (b) Baumann,
H.; Buhler, M.; Fochem, H.; Hirsinger, F.; Zoebelein; H.; Falbe, J. Angew.
Chem., Int. Ed. Eng. 1988, 27, 41.
(28) (a) Morris, L. J.; Harris, R. V.; Kelly, W.; James, A. T. Biochem.
J. 1968, 109, 673. (b) Morris, L. J. Biochem . Biophys. Res. Commun. 1964,
29, 311. (c) Moreau, R. A.; Stumpf, P. K. Plant Physiol. 1981,67, 672. (d)
van de Loo, F. J.; Broun, P.; Turner, S.; Somerville, C. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 6743 and references therein.
(20) Collins, J. R.; Camper, D. L.; Loew, G. H. J. Am. Chem. Soc. 1991,
113, 2736.
(21) Akhtar, M.; Wright, J. N. Nat. Prod. Rep. 1991, 8, 527.
(22) We consider pathway c to be the least likely route to olefin because
the available evidence23 suggests that alcohols are not discrete intermediates
in fatty acid desaturation.