J. Am. Chem. Soc. 1996, 118, 8499-8500
8499
Scheme 1. Formation of Multiple Sesquiterpenes by
Cyclization of Farnesyl Diphosphate (1) by Mutant
Trichodiene Synthases
Enzymatic Formation of Isochamigrene, a Novel
Sesquiterpene, by Alteration of the Aspartate-Rich
Region of Trichodiene Synthase
David E. Cane,* Qun Xue, and James E. Van Epp
Department of Chemistry, Box H
Brown UniVersity
ProVidence, Rhode Island 02912
Youla S. Tsantrizos
Department of Chemistry and Biochemistry
Concordia UniVersity
1455, de MaisonneuVe BouleVard West
Montreal, Que´bec H3G 1M8 Canada
ReceiVed June 6, 1995
Sesquiterpene cyclases are among nature’s most versatile and
intriguing catalysts. Together these enzymes are responsible
for the formation of more than 200 distinct sesquiterpenes carbon
skeletons in plants and microorganisms. Each individual cyclase
is capable of converting the universal acyclic precursor farnesyl
diphosphate (FPP, 1) to a distinct sesquiterpene.1 Over the last
30 years, extensive studies have established a unified model
for these complex transformations based on a common mech-
anism involving ionization of the allylic pyrophosphate ester
followed by a precise sequence of intramolecular electrophilic
addition reactions.2 A major determinant which controls
molecular diversity in product structure and stereochemistry is
believed to be the precise folding of the FPP substrate at the
cyclase active site. Although many of the mechanistic and
stereochemical details of these mechanisms have been verified
by a wealth of isotopic labeling experiments,2,3 little is known
about the active site of any cyclase or the manner in which a
sesquiterpene synthase imposes a particular conformation on
its highly lipophilic substrate, precisely controls the resulting
cascade of electrophilic cyclizations and carbon skeletal rear-
rangement, and ultimately quenches the positive charge.
Trichodiene synthase catalyzes the conversion of FPP to
trichodiene (2), the parent hydrocarbon of the trichothecane
family of antibiotics and mycotoxins.4 The cyclase from
Fusarium sporotrichioides, a homodimer of 45 kDa subunit,
has been cloned5a and overexpressed in Escherichia coli.5b
Experiments with labeled substrates,4a,b,6 as well as with
substrate7 and intermediate8 analogs, have provided support for
the cyclization mechanism illustrated in Scheme 1 (pathway a)
in which FPP, folded in the manner shown, undergoes initial
ionization and rearrangement to (3R)-nerolidyl diphosphate
[(3R)-NPP, 3]. Rotation around the 2,3-bond of 3 followed by
reionization to generate a cisoid allylic cation-pyrophosphate
anion pair allows cyclization to yield the bisabolyl cation 4.
Further cyclization of 4 followed by a 1,4-hydride shift gives
5, from which a consecutive pair of 1,2-methyl migrations and
a final deprotonation result in the formation of trichodiene (2).
None of the proposed enzyme-bound intermediates have been
directly observed with the wild-type enzyme, which produces
trichodiene as the sole product.
Recent studies have been focused on modification of the
enzyme active site by site-directed mutagenesis.9 We have
reported that various mutants of trichodiene synthase modified
in an apparent active-site, arginine-rich domain produce mixtures
of sesquiterpene hydrocarbons, including (-)-(Z)-R-bisabolene
(6), â-bisabolene (7), and cuprenene (8), in addition to the
natural cyclization product trichodiene (2).10 Each of the
aberrant cyclization products could in principle result from
premature deprotonation of the normal cationic cyclization
intermediates (Scheme 1, pathways b, c, and d). More recently
we have extended these studies to mutants altered in a highly
conserved aspartate-rich domain,11 DDSKD, believed to mediate
substrate binding, and possibly ionization, by chelation of the
required divalent Mg2+ ion.12 In fact, the D98E, D99E, and
D102E mutants generated varying proportions of as many as
five anomalous sesquiterpene hydrocarbons in addition to
trichodiene (2), including 6, 7, and 8 as well as â-farnesene (9)
(1) Croteau, R.; Cane, D. E. In Methods in Enzymology. Steroids and
Isoprenoids; Law, J. H., Rilling, H. C., Eds.; Academic Press: New York,
1985; Vol. 110, pp 383-405.
(2) Cane, D. E. Chem. ReV. 1990, 9, 1089-1103. Cane, D. E. Acc.
Chem. Res. 1985, 18, 220-226.
(3) Cane, D. E. In Biosynthesis of Isoprenoid Compounds; Porter, J. W.,
Spurgeon, S. L., Eds.; J. Wiley & Sons: New York, 1981; Vol. 1, pp 283-
374.
(4) (a) Evans, R.; Hanson, J. R. J. Chem. Soc., Perkin Trans. 1 1976,
326-329. (b) Cane, D. E.; Swanson, S.; Murthy, P. P. N. J. Am. Chem.
Soc. 1981, 103, 2136-2138. (c) Hohn, T. M.; Beremand, M. N. Appl.
EnViron. Microbiol. 1989, 55, 1500-1503. (d) Hohn, T. M.; VanMiddles-
worth, F. Arch. Biochem. Biophys. 1986, 251, 756-761.
(5) (a) Hohn, T. M.; Plattner, R. D. Gene 1989, 79, 131-138. Hohn,
T. M.; Plattner, R. D. Arch. Biochem. Biophys. 1989, 275, 92-97. (c) Cane,
D. E.; Wu, Z.; Oliver, J. S.; Hohn, T. M. Arch. Biochem. Biophys. 1993,
300, 416-422.
(6) Cane, D. E.; Ha, H.; Pargellis, C.; Waldmeier, F.; Swanson, S.;
Murthy, P. P. N. Bioorganic Chem. 1985, 13, 246-265. Cane, D. E.; Ha,
H. J. Am. Chem. Soc. 1988, 100, 6865-6870.
(9) Cane, D. E.; Shim, J. H.; Xue, Q.; Fitzsimons, B. C.; Hohn, T. M.
Biochemistry 1995, 34, 2480-2488.
(7) Cane, D. E.; Yang, G.; Xue, Q.; Shim, J. H. Biochemistry 1995, 34,
2471-2479.
(10) Cane, D. E.; Xue, Q. J. Am. Chem. Soc. 1996, 118, 1563-1564.
(11) (a) Ashby, M. N.; Edwards, P. A. J. Biol. Chem. 1990, 265, 13157-
13164. (b) Joly, A.; Edwards, P. A. J. Biol. Chem. 1993, 268, 26983-
26989. (c) Song, L.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
3044-3048.
(8) (a) Cane, D. E.; Pawlak, J. L.; Horak, R. M.; Hohn, T. M.
Biochemistry 1990, 29, 5476-5490. (b) Cane, D. E.; Yang, G. J. Org.
Chem. 1994, 59, 5794-5798. (c) Cane, D. E.; Yang, G.; Coates, R. M.;
Pyun, H.; Hohn, T. M. J. Org. Chem. 1992, 57, 3454-3462.
(12) Cane, D. E.; Xue, Q.; Fitzsimons, B. C. Biochemistry In press.
S0002-7863(96)01897-5 CCC: $12.00 © 1996 American Chemical Society