A common mechanism for branching, cyclopropanation, and cyclobutanation reactions in the isoprenoid biosynthetic pathway

Article abstract of DOI:10.1021/ja0771282

Four reactions - chain elongation, cyclopropanation, branching, and cyclobutanation - are used in nature to join isoprenoid units for construction of the carbon skeletons for over 55 000 naturally occurring isoprenoid compounds. Those molecules produced by chain elongation have head-to-tail (regular) carbon skeletons, while those from cyclopropanation, branching, or cyclobutanation have non-head-to-tail (irregular) skeletons. Although wild type enzymes have not been identified for the branching and cyclobutanation reactions, chimeric proteins constructed from farnesyl diphosphate synthase (chain elongation) and chrysanthemyl diphosphate synthase (cyclopropanation) catalyze all four of the known isoprenoid coupling reactions to give a mixture of geranyl diphosphate (chain elongation), chrysanthemyl diphosphate (cyclopropanation), lavandulyl diphosphate (branching), and maconelliyl and planococcyl diphosphate (cyclobutanation). Replacement of the hydrogen atoms at C1 or C2 or hydrogen atoms in the methyl groups of dimethylallyl diphosphate by deuterium alters the distribution of the cyclopropanation, branching, and cyclobutanation products through primary and secondary kinetic isotope effects on the partitioning steps of common carbocationic intermediates. These experiments establish the sequence in which the intermediates are formed and indicate that enzyme-mediated control of the carbocationic rearrangement and elimination steps determines the distribution of products.

Full text of DOI:10.1021/ja0771282

Published on Web 01/17/2008
A Common Mechanism for Branching, Cyclopropanation, and
Cyclobutanation Reactions in the Isoprenoid Biosynthetic
Pathway
Hirekodathakallu V. Thulasiram,† Hans K. Erickson,‡ and C. Dale Poulter*
Department of Chemistry, UniVersity of Utah, RM 2020, 315 South 1400 East,
Salt Lake City, Utah 84112
Received September 14, 2007; E-mail: poulter@chemistry.utah.edu
Abstract: Four reactionsschain elongation, cyclopropanation, branching, and cyclobutanationsare used
in nature to join isoprenoid units for construction of the carbon skeletons for over 55 000 naturally occurring
isoprenoid compounds. Those molecules produced by chain elongation have head-to-tail (regular) carbon
skeletons, while those from cyclopropanation, branching, or cyclobutanation have non-head-to-tail (irregular)
skeletons. Although wild type enzymes have not been identified for the branching and cyclobutanation
reactions, chimeric proteins constructed from farnesyl diphosphate synthase (chain elongation) and
chrysanthemyl diphosphate synthase (cyclopropanation) catalyze all four of the known isoprenoid coupling
reactions to give a mixture of geranyl diphosphate (chain elongation), chrysanthemyl diphosphate
(cyclopropanation), lavandulyl diphosphate (branching), and maconelliyl and planococcyl diphosphate
(cyclobutanation). Replacement of the hydrogen atoms at C1 or C2 or hydrogen atoms in the methyl groups
of dimethylallyl diphosphate by deuterium alters the distribution of the cyclopropanation, branching, and
cyclobutanation products through primary and secondary kinetic isotope effects on the partitioning steps
of common carbocationic intermediates. These experiments establish the sequence in which the
intermediates are formed and indicate that enzyme-mediated control of the carbocationic rearrangement
and elimination steps determines the distribution of products.
Scheme 1. Four Fundamental Coupling Reactions
The biosynthesis of isoprenoid compounds is nature’s most
chemically diverse pathway, with over 55 000 known metabo-
lites. Many of these molecules fill key metabolic and structural
roles in cells, and the genes encoding isoprenoid enzymes appear
in all forms of life except for a few symbiotic bacteria.1 Better
known groups of isoprenoid compounds include sterols (hor-
mones, membrane components), carotenoids (visual pigments,
photoprotective agents), prenylated proteins (membrane struc-
ture, cell signaling), dolichols (glycoprotein synthesis, bacterial
cell wall synthesis), monoterpenes (insect sex pheromones,
fragrances), and sesquiterpenes (plant defensive agents). Only
the four biosynthetic reactions (see Scheme 1)schain elongation,
cyclopropanation, branching, and cyclobutanationsare used to
couple isoprenoid intermediates to construct more complex
carbon skeletons.2 The most common reaction, chain elongation,
couples isopentenyl diphosphate (IPP) with an allylic isoprenoid
diphosphate to form the 1′-4 linkage characteristic of head-
to-tail or “regular” isoprenoids.3 The enzymes that catalyze 1′-4
coupling are ubiquitous. The other three reactions produce non-
head-to-tail or “irregular” carbon skeletons. A c1′-2-3 cyclo-
propanation reaction is the first pathway-specific step in the
biosynthesis of sterols and carotenoids found in all eukaryotes
and archaea and in some bacteria.4 A similar reaction produces
the cyclopropane-containing skeleton found in the pyrethrin
family of naturally occurring insecticides found in chrysanthe-
mums.5 Metabolites from 1′-2 branching, which are much less
common, are found in a limited number of plants.6,7 The only
documented metabolites from c1′-2-3-2′ cyclobutanation are
mealy bug mating pheromones.8,9
† Present address: Division of Organic Chemistry, National Chemical
Laboratory, Pune 411008, India.
‡ Present address: ImmunoGen Incorporated, 128 Sidney Street, Cam-
bridge, MA 02139, USA.
(4) Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77.
(5) Godin, P. J.; Thain, E. M. Proc. Chem. Soc. 1961, 9, 452.
(6) Gunawardena, K.; Rivera, S. B.; Epstein, W. W. Phytochemistry 2002, 59,
197-203.
(7) Giner, J.-L.; Berkowitz, J. D.; Andersson, T. J. Nat. Prod. 2000, 63, 267-
269.
(1) Perez-Brocal, V.; Gil, R.; Ramos, S.; Lamelas, A.; Postigo, M.; Michelena,
J. M.; Silva, F. J.; Moya, A.; Latorre, A. Science 2006, 314, 312-313.
(2) Thulasiram, H. V.; Erickson, H. K.; Poulter, C. D. Science 2007, 316, 73-
76.
(3) Poulter, C. D. Phytochem. ReV. 2006, 5, 17-26.
9
1966
J. AM. CHEM. SOC. 2008, 130, 1966-1971
10.1021/ja0771282 CCC: $40.75 © 2008 American Chemical Society

Reactions in the Isoprenoid Biosynthetic Pathway
A R T I C L E S
Farnesyl diphosphate synthase (FPPase) is the most widely
distributed of the chain elongation enzymes. It catalyzes two
reactionssthe sequential addition of the hydrocarbon units of
dimethylallyl diphosphate (DMAPP, C5) and geranyl diphos-
phate (GPP, C10) to isopentenyl diphosphate (IPP, C5) to give
farnesyl diphosphate (FPP, C15). Because of its central role in
isoprenoid biosynthesis, FPP synthase has served as a platform
for studying the mechanism of the chain elongation reaction
and structure/function relationships for chain elongation en-
zymes. The isoprenoid units in DMAPP and GPP are joined to
IPP by a dissociative electrophilic alkylation of the double bond
in IPP by allylic cations generated from the allylic diphosphate
substrates.10 FPPase has a distinctive all R-helical fold, first seen
in the X-ray structure of the avian enzyme.11 In the FPP
synthase-substrate complex, the diphosphate moiety of IPP is
surrounded by six molecules of water, while the diphosphate
group in the allylic substrates interacts directly with three Mg2+
atoms and positively charged side chains of arginine and lysine
residues.12 This mode of substrate binding activates the allylic
substrate for the electrophilic alkylation while not activating
the allylic product formed during each step chain elongation.
The iterative addition of a growing allylic chain to IPP requires
relocation of the allylic product to the allylic substrate binding
site during each step of chain elongation. The ultimate length
of the growing isoprenoid chain is determined by the depth of
the binding pocket for the allylic substrate and can be altered
by site-directed mutagenesis to change the size of the side chains
for amino acids in the binding pocket.13,14
The R-helical fold originally seen in FPPase is found in a
superfamily of enzymes in the isoprenoid biosynthetic pathway
whose members also catalyze cyclopropanation in the sterol and
carotenoid pathways15 and the cyclization reactions seen during
biosynthesis of mono-, sesqui-, and diterpenes.16 The fullest
expression of structural diversity in the isoprenoid pathway is
seen in the constituents of the essential oils of plants. One
species, Artemisia tridentata ssp. spiciformis (snowfield sage-
brush) is unique among all organisms in that its essential oil
contains monoterpenes whose structures represent all known
irregular isoprenoid skeletons, except for those of the mealy
bug mating pheromones and membrane lipid tetraethers in
archaea.6 We recently isolated and cloned the genes for FPPase
and CPPase from A. tridentata ssp. spiciformis.17 The encoded
proteins have an extraordinarily high level of sequence similar-
ity. Chimeric enzymes constructed by replacing amino acids,
beginning at the N-terminus, of one protein with increasing
Figure 1. Deuterium labeled substrates.
stretches of sequence from the other show a progression of
activities from 1′-4 elongation, through 1′-2 branching and
c1′-2′-3′-2 cyclobutanation, to c1′-2-3 cyclopropanation.2
Three of these proteins catalyzed all four of the coupling
reactions, suggesting that chain elongation, branching, cyclo-
propanation, and cyclobutanation reactions proceed by similar
chemical mechanisms. We now describe experiments demon-
strating isotopically induced changes in the flux for the
cyclopropanation, branching, and cyclobutanation reactions,
demonstrating that these metabolites are formed from common
carbocationic intermediates through a well-defined sequence of
rearrangements and provide insight about how the biosynthetic
enzymes can optimize synthesis of an individual structure.
Results
Non-Head-to-Tail Products. DMAPP and the deuterated
derivatives shown in Figure 1 were incubated with A. tridentata
ssp. spiciformis CPP synthase and the c98f chimera, constructed
by replacing the first 98 residues in A. tridentata ssp. spiciformis
FPP synthase with the corresponding sequence from A. triden-
tata CPP synthase.2 The C10 diphosphate-containing products
were hydrolyzed with alkaline phosphatase, and the alcohols
were extracted with methyl tert-butyl ether. The extracts were
analyzed by gas chromatography and mass spectrometry. We
previously reported that incubation of DMAPP with CPP
synthase, followed by alkaline phosphatase, gave a 33:17
mixture of (1R,3R)-chyrsanthemol ((1R,3R-COH) and (R)-
lavandulol ((R)-LOH), along with a small amount of (R)-
maconelliol ((R)-MOH) (see Scheme 2). A similar experiment
with the c98f chimera gave a 6:5 mixture of (R)-LOH and (R)-
MOH and a small amount of planococcyl/alcohol (POH).
Related structures were formed when chrysanthemyl diphos-
phate (CPP) synthase and the c98f chimera were incubated with
the deuterium labeled DMAPPs (Figure 1). The monoterpene
alcohols from incubation of (R)- and (S)-[1-2H]DMAPP with
CPP synthase and mass spectral fragmentation patterns for
diagnostic peaks are shown in Scheme 3. COH and LOH from
the R-enantiomer contained two deuterium atoms as seen by
the shift of the molecular ion from m/z 154 for the unlabeled
alcohols to m/z 156 (Figures S7 and S8). A shift of the m/z 123
fragment from loss of the hydroxymethyl unit to m/z 124 in the
mass spectra of both alcohols and a shift in the m/z 69 fragment
to m/z 70 in the mass spectrum of LOH indicate that the labels
were not scrambled. In contrast, incubation with (S)-[1-2H]-
DMAPP gave COH containing a single deuterium due to
elimination of deuterium at C3 when the cyclopropane ring was
formed, as established by a molecular ion at m/z 155 and a peak
at m/z 123, showing that the deuterium atom was in the
hydroxymethyl unit (Figure S7). Thus, the pro-S hydrogen is
selectively removed from C1 of the dimethylallyl unit that is
(8) Zhang, A.; Amalin, D.; Shirali, S.; Serrano, M. S.; Franqui, R. A.; Oliver,
J. E.; Klun, J. A.; Aldrich, J. R.; Meyerdirk, D. E.; Lapointe, S. L. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 9601-9606.
(9) Bierl-Leonhardt, B. A.; Moreno, D. S.; Schwarz, M.; Fargerlund, J.;
Plimmer, J. R. Tetrahedron Lett. 1981, 389-392.
(10) Dolence, J. M.; Poulter, C. D. Electrophilic Alkylations, Isomerizations,
and Rearrangements. In ComprehensiVe Natural Products Chemistry; Meth-
Cohn, O., Ed.; Elsevier: Oxford, 1998; Vol. 5, pp 18473-18500.
(11) Tarshis, L. C.; Yan, M.; Poulter, C. D.; Sacchettini, J. C. Biochemistry
1994, 33, 10871-10877.
(12) Hosfield, D. J.; Zhang, Y.; Dougan, D. R.; Broun, A.; Tari, L. W.; Swanson,
R. V.; Finn, J. J. Biol. Chem. 2004, 279, 8526-8529.
(13) Tarshis, L. C.; Proteau, P. J.; Kellogg, B. A.; Sacchettini, J. C.; Poulter, C.
D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 15018-15023.
(14) Fernandez, S. M.; Kellogg, B. A.; Poulter, C. D. Biochemistry 2000, 39,
15316-17.
(15) Pandit, J.; Danley, D. E.; Schulte, G. K.; Mazzallupo, S.; Pauly, T. A.;
Hayward, C. M.; Hamaka, E. S.; Thompson, J. F.; Harwood, H. J. J. Biol.
Chem. 2000, 275, 30610-30617.
(16) Christiansen, D. W. Chem. ReV. 2006, 106, 3412-3442.
(17) Hemmerlin, A.; Rivera, S. B.; Erickson, H.; Poulter, C. D. J. Biol. Chem.
2003, 34, 32132-32140.
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1967

A R T I C L E S
Thulasiram et al.
Scheme 2. Non-Head-to-Tail Alcohols after Incubation of DMAPP
with CPP Synthase or c98f Followed by Hydrolysis with Alkaline
Phosphatase
Scheme 4. Alcohols from Incubation of (E)- and
(Z)-[4,4,4-2H3]DMAPP and [4,4,4,5,5,5-2H6]DMAPP with CPP
Synthase
Scheme 3. Alcohols from Incubation of (R)- and (S)-[1-2H]DMAPP
with CPP Synthase
m/z 154 to m/z 160, and the peak at m/z 123 from loss of the
hydroxymethyl fragment shifts to m/z 129 (Figures S9 and S12).
The molecular ion of COH from (CD3)2-DMAPP is at m/z 166,
and loss of the hydroxymethyl fragment gives a peak at m/z
135. The patterns seen for LOH are more complex. LOH from
(E)- or (Z)-CD3-DMAPP gives a molecular ion at m/z 159
(C10H13D5O) or 160 (C10H12D6O), depending on whether a
hydrogen or deuterium atom is eliminated to give the disubsti-
tuted double bond (Figures S12, S13, S18, and S19). The pattern
was easier to discern in the more intense peaks at m/z 128
(C9H10D5) and m/z 129 (C9H9D6) after loss of the hydroxy-
methyl fragment. The relative intensities of the m/z 159/160
and 128/129 peaks varied in scans taken as LOH eluted from
the GC column, with the higher mass fragments appearing
earlier in the elution. The GC peak for LOH derived from CD3-
DMAPP had a shoulder, indicating that the C10H13D5O and
C10H12D6O forms of LOH had slightly different retention times
with the C10H12D6O species eluting first (see Figure 2). The
relative amount of C10H13D5O and C10H12D6O was determined
by curve-fitting a plot of peak intensity versus retention time
to a model consisting of two overlapping Gaussian peaks in a
71:29 ratio.
inserted into the C2-C3 double bond of DMAPP. This is the
same stereochemistry that was observed for formation of
presqualene diphosphate and prephytoene diphosphate during
sterol18 and carotenoid biosynthesis.19 The mass spectrum of
LOH from (S)-[1H]DMAPP was identical to the one obtained
for LOH produced from the R-enantiomer (Figure S8 and S10).
The products from the three CD3-labeled DMAPP derivatives
and characteristic fragments are shown in Scheme 4. The
molecular ions of COH from (E)- or (Z)-CD3-DMAPP shift from
Product Distributions. The relative amounts of the non-head-
to-tail alcohols from incubations of CPP synthase and the c98f
chimera with DMAPP and several deuterated derivatives,
followed by hydrolysis with alkaline phosphatase, are shown
in Table 1.
Changes in the product ratios are consistent with a combina-
tion of primary and secondary deuterium isotope effects on
partitioning steps that lead to the products. The largest change
(18) Poulter, C. D.; Rilling, H. C. “Conversion of Farnesyl Pyrophosphate to
Squalene.” In Biosynthesis of Isoprenoid Compounds; Porter, J. W., Ed.;
John Wiley & Sons: New York, 1981; Volume 1, Chapter 8, pp 414-
441.
(19) Gregonis, D. E.; Rilling, H. C. Biochemistry 1974, 13, 1538-1542.
9
1968 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Reactions in the Isoprenoid Biosynthetic Pathway
A R T I C L E S
∼1.12-fold isotope effect. The MOH/POH ratio increased to
13.6 for (CD3)2-DMAPP, a 1.21-fold change.
Discussion
The observation that the four reactions used to join isoprenoid
unitsschain elongation, cyclopropanation, branching, and
cyclobutanationsare catalyzed by a single enzyme led us to
propose an integrated mechanism for biosynthesis of these
structures from shared intermediates based on the dissociative
electrophilic alkylation mechanism for chain elongation.2 The
enzymes that catalyze chain elongation and cyclopropanation
have similar folds, indicating that they evolved from a common
ancestor. Although the enzymes that catalyze branching and
cyclobutanation are unknown, the absolute stereochemistries of
the chiral centers in naturally occurring COH, LOH, MOH, and
POH are identical, as are those in the alcohols obtained from
the c98f chimeria of CPP and FPP synthase. These observations
are consistent with a scenario where the enzymes that catalyze
cyclopropanation, branching, and cyclobutanation evolved from
an ancestral chain elongation enzyme through gene duplication/
mutagenesis. Thus, the preferred product for a particular enzyme
would be determined by the relative efficiencies of the rear-
rangement and proton elimination steps in the series of reactions
initiated by alkylation of DMAPP by the dimethylallyl cation
(DMA+) shown in Scheme 5. The absolute stereochemistries
of the products would be dictated by binding interactions within
the structurally related chiral environments of the enzyme’s
active sites. It is, however, possible that cyclopropanation
and branching products are synthesized by parallel pathways
entered independently during alkylation of DMAPP by DMA+
to generate the chrysanthemyl cation (C+) or the lavandulyl
cation (L+). It is also possible that L+ is the initial alkylation
product of DMA+ and DMAPP, and L+ subsequently iso-
merizes to C+. The maconelliyl cation (M+) is most likely
generated by cyclization of L+. Computational and experimental
studies suggest that the difference in free energy between
isomeric protonated cyclopropanes and tertiary carbocations can
be sufficiently low to permit rapid equilibration of the two
species.20,21 Thus, any of the three scenarios mentioned above
are candidates for the mechanism of the biosynthetic reactions.
These issues were resolved by studying the effect of deuterium
substitution on the formation of CPP and LPP (lavandulyl
diphosphate) by CPP synthase and of LPP and MPP (maconel-
liyl diphosphate) by the c68f chimera.
Figure 2. GC trace of C10 alcohols from incubation of DMAPP (a) and
(Z)-CD3-DMAPP (b) with A. tridentata CPP synthase followed by alkaline
phosphatase.
Table 1. Distributions of the Major Products from Incubation of
DMAPP with A. Tridentata ssp. Spiciformis CPP Synthase
(COH/LOH) and the c98f CPP/FPP Synthase Chimera (LOH/MOH
and MOH/POH) after Treatment with Alkaline Phosphatasea
substrate
COH/LOH
LOH/MOH
MOH/POH
DMAPP
3.9 ( 0.1
3.7 ( 0.05
0.81 ( 0.02
6.5 ( 0.3
6.6 ( 0.1
10.8 ( 0.5
3.7 ( 0.1
1.85 ( 0.03
-
-
1.83 ( 0.07
1.80 ( 0.02
1.27 ( 0.02
1.86 ( 0.06
11.2 ( 0.4
-
-
10.5 ( 0.9
10.5 ( 0.5
13.6 ( 1.3
9.9 ( 0.9
(R)-[1-2H]DMAPP
(S)-[1-2H]DMAPP
(Z)-[4,4,4-2H3]DMAPP
(E)-[4,4,4-2H3]DMAPP
[4,4,4,5,5,5-2H6]DMAPP
[2-2H]DMAPP
a Each value is the average from three independent incubations.
in the COH/LOH ratio is a 4.9-fold decrease in [COH/LOH]H/
[COH/LOH]D when DMAPP is replaced by (S)-[1-2H]DMAPP.
Substitution of one of the methyl groups in DMAPP with a
CD3 moiety results in smaller, but significant, increases in the
COH/LOH ratio reflecting isotope effects of [COH/LOH]H/
[COH/LOH]D ) 1.67 and 1.69 for the Z- and E-CD3 derivatives,
respectively. The COH/LOH ratio increased to 10.8 for (CD3)2-
DMAPP, corresponding to [COH/LOH]H/[COH/LOH]D ) 2.77.
Deuterium substitution at C2 of DMAPP did not significantly
alter the COH/LOH ratio.
A similar study was conducted looking at changes in the
LOH/MOH and MOH/POH ratios from incubations of the c98f
chimera with [2-2H]DMAPP and the CD3-DMAPP derivatives.
The LOH/MOH ratios seen for DMAPP, [2-2H]DMAPP, and
the mono CD3 DMAPP derivatives were all within experimental
error, indicating that there was no discernible isotope effects
on the product-forming steps. The ratio decreased from 1.85 to
1.27 for (CD3)2-DMAPP, a 1.46-fold change in the product
forming steps. The MOH/POH ratios for DMAPP and the mono
CD3 DMAPPs were within experimental error. The decrease in
the ratio from 11.2 to 9.9 for [2-2H]DMAPP indicates a small
During biosynthesis of CPP, the pro-S hydrogen (green) is
lost from the C1 position of the electrophilic DMAPP. Incuba-
tion of (S)-[1-2H]DMAPP with CPP synthase, followed by
hydrolytic workup, gives COH with a single deuterium atom
in the hydroxymethyl group; whereas, the deuterium atoms from
both molecules of DMAPP are incorporated into COH derived
from the R-enantiomer. Furthermore, the COH/LOH ratio drops
∼4.9-fold in incubations with (S)-[1-2H]DMAPP relative to the
ratio seen for (R)-[1-2H]DMAPP or unlabeled DMAPP. These
results demonstrate a substantial primary kinetic isotope effect
(KIE) on the partitioning of C+ to CPP (step a) and L+ (step b)
as a result of elimination of deuterium in the CPP product-
(20) Farcasiu, D.; Norton, S. H.; Hancu, D. J. Am. Chem. Soc. 2000, 122, 668-
676.
(21) Walker, G. E.; Kronja, O.; Saunders, M. J. Org. Chem. 2004, 69, 3598-
3601.
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1969

A R T I C L E S
Thulasiram et al.
Scheme 5. A Common Mechanism for the Stereoselective Biosynthesis of Irregular Isoprenoid Compounds with c1′-2-3, 1′-2, and
c1′-2-3-2′ Skeletons from DMAPP with the C1 Hydrogens Labeled: ProR (Red) and ProS (Green)
forming step.22 The magnitude of the KIE indicates that C+ is
the direct precursor of L+.
on the COH/LOH ratio suggests that protonated cyclopropane
C+ is the first intermediate generated upon condensation of
DMA+ with the C2-C3 double bond in DMAPP. The ratio
CPP to LPP is dictated by the relative rates for steps a and b.
This scenario is consistent with the large primary deuterium
KIE seen for step a and the positive â-secondary deuterium
isotope effect for step b. Following rearrangement of C+ to L+,
the tertiary carbocation can lose a proton to give LPP or cyclize
to give M+. Finally, M+ can lose the cyclobutyl proton to give
MPP or a methyl proton to give PPP (planococcyl diphosphate).
The rearrangement (b and d) and elimination (a, c, e, and f)
steps appear to be irreversible.
Formation of cyclopropanation, branching, and cyclobutana-
tion products by deprotonation of the carbocationic intermediates
in the C+ f L+ f M+ cascade of rearrangements provides an
opportunity for facile evolution of enzymes for selective
biosynthesis of related irregular monoterpenes. The selectivity
for synthesis of isoprenoid compounds with a specific carbon
skeleton depends on how C+, L+, and M+ partition between
the options for elimination and rearrangement. The relative rates
of these competitive reactions depend on the ability of an
enzyme to stabilize C+, L+, and M+ by a combination of
charge-charge and charge-dipole interactions between the
carbocations and the enzyme24 and to promote the regioselective
elimination of protons with strategically located bases in the
active site.
This hypothesis is confirmed by a KIE on the COH/LOH
ratio seen in incubations with (Z)- and (E)-CD3-DMAPP and
(CD3)2-DMAPP. The COH/LOH ratios are identical for both
stereoisomers and correspond to a positive secondary deuterium
KIE of 1.67 for the CD3 group, which is independent of its
stereochemistry on the cyclopropane ring in C+. The COH/LOH
ratio for (CD3)2-DMAPP corresponds to an observed secondary
â-deuterium KIE of 2.77 (or 1.67/CD3 group). A positive
secondary â-deuterium KIE is consistent with rate limiting
isomerization of C+ to L+ (step b) where the hybridization of
the methyl-bearing carbon approaches sp2 in the transition
state.23
Mass spectroscopic and gas chromatographic analysis of LOH
obtained from (Z)-CD3-DMAPP indicates an ∼2.4-fold prefer-
ence for elimination of a proton from the CH3 group rather than
a deuterium from the CD3 in step c. This value reflects a
combination of the intrinsic primary KIE and a stereoselective
preference imposed by CPP synthase during the elimination step.
Values ranging from 2.3 to 5.9 have been reported for related
eliminations in reactions catalyzed by monoterpene cyclases.24
The LOH/MOH ratio does not change for incubations with
DMAPP, [2-2H]DMAPP, and (Z)- and (E)-CD3-DMAPP, re-
flecting no detectible change in the relative rates of steps c and
d. However, the ratio decreases ∼1.46-fold when (CD3)2-
DMAPP is the substrate. In this case, mandatory loss of a
deuterium in step c results in a modest primary KIE on the
partitioning of L+ to LPP and M+. Finally, a modest decrease
in the MOH/POH ratio when [2-2H]DMAPP is the substrate is
consistent with a small primary deuterium KIE on step e, while
a small increase in the MOH/POH ratio for (CD3)2-DMAPP is
consistent with a small primary isotope effect of step f.
The mechanism for biosynthesis of the four fundamental
building reactions in the isoprenoid pathway presented in
Scheme 5 is supported by deuterium isotope effects on key
rearrangement and elimination steps. The large isotope effect
Materials and Methods
General. Unlabeled DMAPP25 and deuterium labeled DMAPP26
were prepared as described in the literature. Chrysanthemum cinerari-
aefolium CPP synthase,27 Artemisia tridentata ssp. spiciformis CPP
synthase,18 and the c98f FPP synthase/CPP synthase A. tridentata ssp.
spiciformis chimera2 were purified from Escherichia coli hosts as
previously reported. Authentic samples and mass spectra of (1R,3R)-
chrysanthemol (COH), (R)-lavandulol (LOH), (R)-maconelliol (MOH),
and (1R,3R)-planococcol (POH) were available from a previous study.2
(25) Davisson, V. J.; Woodside, A. B.; Neal, T. R.; Stremler, K. E.; Muehlbacher,
M.; Poulter, C. D. J. Org. Chem. 1986, 51, 4768-4779.
(26) Thulasiram, H. V.; Phan, M. R.; Rivera, B. S.; Poulter C. D. J. Org. Chem.
2006, 71, 1739-1741.
(27) Rivera, S. B.; Swedlund, B. D.; King, G. J.; Bell, R. N.; Hussey, C. E., Jr.;
Shattuck-Eidens, D. M.; Wrobel, W. M.; Peiser, G. D.; Poulter, C. D. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 4373-4378.
(22) Wolfsberg, M. Acc. Chem. Res. 1972, 7, 225-233.
(23) Frisone, G. J.; Thornton, E. R. J. Am. Chem. Soc. 1968, 90, 1211-1215.
(24) Pyun, H.-J.; Coates, R. M.; Wagschal, K. C.; McGeady, P.; Croteau, R. B.
J. Org. Chem. 1993, 58, 3998-4009.
9
1970 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Reactions in the Isoprenoid Biosynthetic Pathway
A R T I C L E S
Enzymatic Incubations and Product Analysis. Enzyme (400 µg)
was added to 35 mM HEPES buffer, pH 7.6, containing 10 mM MgCl2,
5.0 mM â-mercaptoethanol, and 3 mM DMAPP to a final volume of
400 µL. The mixture was incubated for 12 h at 30 °C. The isoprenoid
diphosphate products were hydrolyzed to the corresponding alcohols
by addition of 80 µL of 500 mM glycine buffer, pH 10.5, containing
5 mM ZnCl2 and 80 units of calf alkaline phosphatase (Sigma) followed
by incubation at 37 °C for 2 h. Approximately 0.2 g of NaCl was added,
and the aqueous phase was extracted three times with 400 µL of tert-
butyl methyl ether. The volume of the extract was reduced to ∼50 µL
with a stream of dry nitrogen. A 1 µL portion of the extract was injected
onto a 30 m × 0.32 mm × 0.25 µm 20% â-cyclodextrin capillary GC
column (HP-CHIRAL-20B, J & W Scientific) equilibrated at 60 °C
followed by a temperature gradient from 60 °C to 120 °C at 2 °C/min,
a temperature gradient of 120 °C to 230 °C at 10 °C/min (program 1)
or injection on the column equilibrated at 50 °C with 3 min at 50 °C
followed by a temperature gradient from 50 °C to 120 °C at 2 °C/min
and 120 °C to 230 °C at 10 °C/min (program 2). Helium was used as
the carrier gas at a flow rate of 1 mL/min. Analysis by GC/MS was
performed under similar conditions using a 30 m × 0.25 mm × 0.25
µm 20% â-cyclodextrin capillary column (HP-CHIRAL-20B, J & W
Scientific) at a flow rate of 1 mL of He/min. The structures of the
products were verified by co-injection with authentic samples and by
mass spectrometry (see Figures S1-S29).2
Acknowledgment. We thank Dr. Aijun Zhang for providing
an authentic sample of (R)-maconelliol and Dr. Joel Harris for
help with analysis of overlapping GC peaks. This work was
supported by NIH Grant GM21328.
Supporting Information Available: GC traces for COH,
LOH, MOH, and POH. Mass spectra for COH, LOH, MOH,
and POH and deuterium-containing derivatives.
JA0771282
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 1971