Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis

Article abstract of DOI:10.1021/ja026058q

Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in k_cat and k_cat/K_m. By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg²⁺-binding aspartate-rich domain, ⁸⁰DDLFD, resulted in reduction in the k_cat/K_m for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a k_cat/K_m which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product β-caryophyllene (9) (9%). Finally, the F77Y mutant had a k_cat/K_m which was reduced by 20-fold compared to that of the wild-type synthase.

Full text of DOI:10.1021/ja026058q

Published on Web 06/08/2002
Pentalenene Synthase. Analysis of Active Site Residues by
Site-Directed Mutagenesis
†
†
§
‡
Myriam Seemann, Guangzhi Zhai, Jan-Willem de Kraker, Chiana M. Paschall,
‡
,†
David W. Christianson, and David E. Cane*
Contribution from the Department of Chemistry, Box H, Brown UniVersity,
ProVidence, Rhode Island 02912-9108, Department of Organic Chemistry,
Wageningen Agricultural UniVersity, Dreijenplein 8, 6703 HB Wageningen, The Netherlands,
Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands, and
Roy and Diana Vagelos Laboratories, Department of Chemistry, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104-6323
Received February 28, 2002
Abstract: Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene
synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying
proportions of (+)-germacrene A (7) with relatively minor changes in kcat and kcat/K
m
. By contrast, single
H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2).
2+
Mutation to glutamate of each of the three aspartate residues in the Mg -binding aspartate-rich domain,
80
m
DDLFD, resulted in reduction in the kcat/K for farnesyl diphosphate and formation of varying proportions
of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene
synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not
simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and
N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant
had a kcat/K
pentalenene (2) (91%) and the aberrant cyclization product â-caryophyllene (9) (9%). Finally, the F77Y
mutant had a kcat/K which was reduced by 20-fold compared to that of the wild-type synthase.
m
which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of
m
Sesquiterpene synthases are a remarkably versatile family of
enzymes that catalyze the cyclization of a single acyclic
precursor, farnesyl diphosphate (1, FPP), to any of 300 known
Scheme 1
1
sesquiterpene hydrocarbons or alcohols. The closely related
monoterpene synthases cyclize geranyl diphosphate (GPP), the
C10 acyclic homologue of FPP, to a variety of monocyclic and
bicylic monoterpenes, using variations of the same ionization-
2
cyclization mechanism. The enzymes do not require any organic
2
+
or inorganic cofactor other than a divalent cation, with Mg
or, in some cases, Mn2+ usually being preferred. The proteins
themselves are usually monomers, or occasional homodimers,
with subunit molecular weights of 40-65 kDa. Each multistep
cyclization reaction normally takes place within a single active
site without release of free intermediates.
3
phate (1) to pentalenene (2), the parent sesquiterpene hydro-
4
carbon of the pentalenolactone antibiotics, a family of me-
tabolites which have been isolated from several species of
Streptomycetes (Scheme 1). The enzyme from Streptomyces
UC5319, a monomer of 38 kDa, has been purified to homoge-
5
Pentalenene synthase is a typical sesquiterpene synthase that
2+
catalyzes the Mg -dependent cyclization of farnesyl diphos-
6
neity, cloned, and overexpressed in Escherichia coli. The
mechanism of the cyclization, which has been extensively
studied using a variety of stereospecifically labeled forms of
*
To whom correspondence should be addressed. E-mail: David_Cane@
brown.edu.
†
Brown University.
Wageningen Agricultural University and Plant Research International.
University of Pennsylvania.
§
‡
(
3) Cane, D. E.; Tillman, A. M. J. Am. Chem. Soc. 1983, 105, 122-124. Cane,
D. E.; Abell, C.; Tillman, A. M. Bioorg. Chem. 1984, 12, 312-328.
(
1) Cane, D. E. In Isoprenoids Including Carotenoids and Steroids; Cane, D.
E., Ed. (Volume 2 in ComprehensiVe Natural Products Chemistry; Barton,
D, Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Elsevier: Oxford,
(4) Seto, H.; Yonehara, H. J. Antibiot. 1980, 33, 92-93.
(5) (a) Martin, D. G.; Slomp, G.; Mizsak, S.; Duchamp, D. J.; Chidester, C.
G. Tetrahedron Lett. 1972, 4901-4904. (b) For examples and leading
references, see: Cane, D. E.; Sohng, J. K.; Williard, P. G. J. Org. Chem.
1992, 57, 844-852.
1
999; pp 155-215.
(
2) Croteau, R. B. In Isoprenoids Including Carotenoids and Steroids; Cane,
D. E., Ed. (Volume 2 in ComprehensiVe Natural Products Chemistry;
Barton, D, Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Else-
vier: Oxford, 1999; pp 97-153.
(6) Cane, D. E.; Sohng, J. K.; Lamberson, C. R.; Rudnicki, S. M.; Wu, Z.;
Lloyd, M. D.; Oliver, J. S.; Hubbard, B. R. Biochemistry 1994, 33, 5846-
5857.
10.1021/ja026058q CCC: $22.00 © 2002 American Chemical Society
J. AM. CHEM. SOC. 2002, 124, 7681-7689
9
7681

A R T I C L E S
Seemann et al.
Figure 1. (A) Crossed-eye stereoview of pentalenene synthase active site, illustrating key active site residues. Polar residues at the top are believed to be
2
+
involved in binding of Mg and the pyrophosphate moiety of the FPP substrate, while aromatic residues deeper in the active site cleft provide a hydrophobic
surface for the farnesyl residue and stabilize carbocation intermediates. View is from the side, with opening of active site at the top of the figure. (Generated
38
39
by Swiss Pdb-Viewer. ) (B) Electrostatic surface of pentalenene synthase generated using GRASP. Red regions indicate negative charge, and blue indicates
positive charge. View is from the top of the active site cavity, with the aspartate-rich domain on the right.
FPP,3,7 is believed to involve initial ionization of the pyrophos-
phate moiety followed by attack of the resulting allylic cation
at C-11 of the distal double bond to give the humulyl cation 3
This conserved topology, which consists entirely of R-helices
connected by short peptide loops, has been termed the class I
8
c
R-helical terpenoid cyclase fold. Interestingly, the same fold
10
(Scheme 1). Rearrangement of 3 to the isomeric humulyl cation
is also observed for avian farnesyl diphosphate synthase and
1
1
4
, either by a 1,2-hydride shift or by a deprotonation-
human squalene synthase, two enzymes which catalyze
mechanistically analogous reactions of allylic diphosphate
substrates. The active sites of all of these enzymes consist of a
conical and hydrophobic active site cleft, flanked by six or seven
helices and lined with an unusual number of aromatic and
nonpolar amino acid residues (Figure 1A). In the case of
pentalenene synthase, the cleft is approximately 9 Å wide and
reprotonation sequence, is followed by cyclization to the seco-
illudyl cation 5. Hydride shift, cyclization, and stereospecific
removal of the H-7re proton from the penultimate tricyclic
intermediate 6 yields pentalenene. The cyclization is prototypical
of all sesquiterpene cyclizations.
With the availability of numerous cloned sesquiterpene and
monoterpene synthases, the role of each cyclase in catalyzing
the formation of its characteristic terpenoid product has been
the object of considerable speculation and intensive study. In
each case, the cyclase protein is believed to bind and fold the
acyclic substrate FPP (or GPP) in a specific conformation and
to initiate cyclization by ionization of the allylic diphosphate
8
a
15 Å deep. The aromatic side chains are thought to play a
critical role in shaping the active site and specifically stabilizing
the succession of carbocationic intermediates that are generated
1
2
in the course of each cyclization. Stabilization of the carbo-
cations is further enhanced by the negative electrostatic charge
of the interior surface of the active site (Figure 1B). Near the
upper edge of the active site cavity of each synthase, located at
the C-terminal end of an R-helix, is a universally conserved
aspartate-rich motif, DDXXD, that has been implicated in the
2+
moiety, assisted by Mg or another divalent cation. Crystal
structures of four different sesquiterpene cyclasesspentalenene
synthase from the Gram-positive bacterium Streptomyces UC5319,
trichodiene synthase and aristolochene synthase from the
filamentous fungi Fusarium sporotrichioides and Penicillium
roqueforti, respectively, and epi-aristolochene synthase from the
binding of the pyrophosphate moiety of the substrate through
1,2,10,13,14
chelation of the requisite divalent cation.
On the
8
tobacco plant, Nicotiana tabacum shave indicated a remarkably
(9) Although sesquiterpene synthases of microbial origin generally show no
significant overall sequence similarity either to one another or to any other
class of proteins (except for orthologs biosynthesizing the same sesqui-
terpene product), the dozens of known monoterpene, sesquiterpene, and
diterpene synthases from plant sources have strongly conserved primary
amino acid sequences as well as a common intron organization and exon
size, consistent with a common evolutionary origin (cf.: Trapp, S. C.;
Croteau, R. B. Genetics 2001, 158, 811-832.) It is highly likely, therefore,
that all such plant terpene synthases will have the same topology as epi-
aristolochene synthase.
high degree of conservation in the three-dimensional structures
of all synthases, despite an essentially complete lack of overall
amino acid sequence similarity among any of these proteins.
9
(
7) Cane, D. E.; Oliver, J. S.; Harrison, P. H. M.; Abell, C.; Hubbard, B. R.;
Kane, C. T.; Lattman, R. J. Am. Chem. Soc. 1990, 112, 4513-4524. Cane,
D. E.; Weiner, S. W. Can. J. Chem. 1994, 72, 118-127. Cane, D. E.;
Abell, C.; Harrison, P. H.; Hubbard, B. R.; Kane, C. T.; Lattman, R.; Oliver,
J. S.; Weiner, S. W. Philos. Trans. R. Soc. B 1991, 332, 123-129.
8) (a) Pentalenene synthase: Lesburg, C. A.; Zhai, G.; Cane, D. E.;
Christianson, D. W. Science 1997, 277, 1820-1824. (b) Trichodiene
synthase: Rynkiewicz, M. J.; Cane, D. E.; Christianson, D. W. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 13543-13548. Rynkiewicz, M. J.; Cane, D.
E.; Christianson, D. W. Biochemistry 2002, 41, 1732-1741. (c) Aris-
tolochene synthase: Caruthers, J. M.; Kang, I.; Rynkiewics, M. J.; Cane,
D. E.; Christianson, D. J. Biol. Chem 2000, 275, 25533-25539. (d) Epi-
aristolochene synthase: Starks, C. M.; Back, K.; Chappell, J.; Noel, J. P.
Science 1997, 277, 1815-1820.
(10) Tarshis, L. C.; Yan, M. J.; Poulter, C. D.; Sacchettini, J. C. Biochemistry
1994, 33, 10871-10877. Tarshis, L. C.; Proteau, P. J.; Kellogg, B. A.;
Sacchetini, J. C.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
15018-15203.
(
(11) Pandit, J.; Danley, D. E.; Schulte, G. K.; Mazzalupo, S.; Pauly, T. A.;
Hayward, C. M.; Hamanaka, E. S.; Thompson, J. F.; Harwood, H. J., Jr. J.
Biol. Chem. 2000, 275, 30610-30617.
(12) (a) Dougherty, D. A. Science 1996, 271, 163-168. (b) Lesburg, C. A.;
Caruthers, J. M.; Paschall, C. M.; Christianson, D. W. Curr. Opin. Struct.
Biol. 1998, 8, 695-703.
(13) Ashby, M. N.; Edwards, P. A. J. Biol. Chem. 1990, 265, 13157-13164.
7682 J. AM. CHEM. SOC.
9
VOL. 124, NO. 26, 2002

Pentalenene Synthase
A R T I C L E S
Table 1. Pentalenene Synthase Mutants
steady-state kinetic parameters
(µM)
0.3
relative proportion of sesquiterpenes (%)
mutant
WT PS
plasmid
k
cat (s-1)
K
m
k
cat/K
m
(s-1 M-1)
2
7
8
10
6
pZW05
pGZ11
pGZ12
pGZ13
pMS11
pMS03
pMS04
pMS05
pMS06
pMS08
PMS09
pGZ14
pGZ17
pMS07
0.3
1.0 × 10
0.6 × 10
0.4 × 10
0.2 × 10
1.2 × 10
4.1 × 10
4.6 × 10
286
100
78
0
0
0
0
0
0
0
0
0
0
0
0
0
6
6
6
4
4
5
H309A
H309S
H309C
H309F
W309F/H309F
W309F
D80E
0.11
0.19
0.25
0.33
1.43
0.61
0.16
6.98
2.6
10
10
13
13
25
7
12
8
0.11
82
0.07
80
7
0.018
0.025
0.074
0.002
0.0065
0.81
84
3
a
62
0
93
0
100
92
0
0
b
D81E
2500
4.5
3
6
D84E
2.28
0.3
0.35 × 10
97
>97
4
F77Y
0.016
5.2 × 10
0
0
0
N219A
N219L
N219D
inactive
inactive 2
0.005
16.6
3.0 × 10
91
0
9
a
The W308F/H309F mutant also produced an additional 13% of a 1:1 mixture of two unidentified hydrocarbons, m/z 204. b The D81E mutant also
produced 3.5% of an unidentified hydrocarbon, m/z 204.
Scheme 2
proportions of two sesquiterpene hydrocarbons which we
identified as germacrene A (7) and protoilludene (8)16 (Scheme
2
). Both of these latter compounds result from derailment of
the normal cyclization cascade, presumably due to relaxed
control over the conformations of the substrate FPP and the
derived carbocationic intermediates in the active sites of the
various H309 mutants. We now report the results of site-directed
mutagenesis of additional active site residues that shed light on
the mechanism of action of pentalenene synthase.
Results
opposite wall is found another highly conserved triad of amino
Active Site Base. Having ruled out H309 as the active site
base, we turned our attention to other plausible amino acid
candidates. Unfortunately, no other nominally basic amino acids
are present in the active site. The various arginine and lysine
residues (R157, R173, K226, and K230) which are present along
the upper region of the active site cavity are expected to be
present in their protonated, positively charged form and are
thought to play a role in binding of the pyrophosphate moiety
of the substrate, rather than as Lewis bases. We therefore had
to consider other, less conventional, possibilities. A tryptophan
residue has previously been suggested as a possible active site
acid side chains, (D/N)DXX(S/T)XXXE, that also plays an
important role in Mg2 binding
+
8b,d,15
(Figure 1A). Despite the
increasing amount of structural information, little is known about
the precise manner by which pentalenene synthase and related
terpene cyclases impose a specific conformation on their acyclic,
lipophilic substrates or trigger the ionization of the allylic
diphosphate, how they mediate the resultant cascade of elec-
trophilic reactions, or how they determine the timing and
stereochemistry of the quenching of the carbocationic intermedi-
ates, either by removal of a specific proton or by capture by an
external nucleophile such as water.
8
d
base for epi-aristolochene synthase. Noting that pentalenene
synthase also has a tryptophan residue (W308) immediately
adjacent to the previously examined H309 (Figure 1A), we
generated two pentalenene synthase mutants in which this
tryptophan was replaced by phenylalanine, W308F, and the
double mutant W308F/H308F (Table 1). Each of these mutant
proteins was purified to homogeneity, and the steady-state
kinetic parameters were determined, as previously described for
the wild-type recombinant synthase. Both mutants retained
substantial pentalenene synthase activity, with kcat lowered by
a factor of only 4-12 and an overall reduction in kcat/Km of
between 2 and 25. Interestingly, the W308F/H309F double
mutant was actually slightly more active than the single H309F
mutant. Unlike the simple H309 mutants, however, neither
W308 mutant produced detectable protoilludene (8). Instead,
the W308F and W308F/H309F mutants generated a mixture of
pentalenene (2) and 7% and 25% of (+)-germacrene A (7),
respectively, with the double mutant producing an additional
In the crystal structure of pentalenene synthase, the only
apparent basic residue in the active site cavity is histidine-309
Figure 1A). Modeling of both FPP and derived intermediates
8a
(
into the active site originally led us to suggest that the imidazole
side chain of H309 might serve as the functional active site
base that mediates the postulated series of deprotonations and
reprotonations, including the final deprotonation that generates
pentalenene. This inference was firmly excluded, however, when
site-directed mutagenesis showed that H309 is not required for
1
6
catalytic activity. Indeed, the H309A, H309C, H309S, and
H309F mutants all retained substantial pentalenene synthase
activity, with relatively minor decreases in kcat ranging from as
little as 3-fold for H309A and H309S to at most ∼17-fold for
the H309F mutant, accompanied by only minor increases in Km
for three of the mutants (Table 1). On the other hand, each of
these mutants was found to produce up to 20% of varying
(
14) Cane, D. E.; Xue, Q.; Fitzsimons, B. C. Biochemistry 1996, 35, 12369-
1
2376.
13% of equal quantities of two as-yet unidentified sesquiterpene
(
(
15) Cane, D. E.; Kang, I. Arch. Biochem. Biophys. 2000, 376, 354-364.
16) Portions of this work dealing with the H309A, H309S, H309C, and H309F
mutants have been published in preliminary form. For details of the
identification of 7 and 8, see: Seemann, M.; Zhai, G.; Umezawa, K.; Cane,
D. J. Am. Chem. Soc. 1999, 121, 591-592.
hydrocarbons. The absolute configuration of the enzymatically
generated (+)-germacrene A (7) was established by means of
its facile Cope rearrangement to (-)-â-elemene (9), a reaction
J. AM. CHEM. SOC.
9
VOL. 124, NO. 26, 2002 7683

A R T I C L E S
Seemann et al.
Figure 2. (A) Difference electron density map calculated with Fourier coefficients |Fo| - |Fc| and phases calculated from the final model less the atoms
of the three indicated residues and all atoms within a 2 Å radius of them, contoured at 2σ. The substituted L219 side chain is indicated along with other
active site residues. (B) Least-squares superposition of N219L pentalenene synthase (yellow) and wild-type pentalenene synthase (white) active sites (orientation
is the same as in panel A). Note that the N219L substitution is accommodated without significant structural changes in the enzyme active site.
Scheme 3
enforcing a specific folding on the substrate, FPP, and possibly
by stabilizing transient cations by dipole-cation interactions.
8d
Structural studies of both epi-aristolochene synthase and
8
b
trichodiene synthase, however, suggest that the carbonyl
oxygen of the side chain amide of N219 in pentalenene synthase
2
+
is part of a highly conserved Mg -binding triad that also
includes the side chain oxygen atoms of serine-223 and
glutamate-227. Accordingly, we generated both the isosteric
N219L mutant and the corresponding N219A mutant (Table 1).
Significantly, each of these mutants was completely inactive,
within the limits of detection of the assay, which could have
which occurs with retention of configuration at C-7, the site of
the 2-propenyl substituent17 (Scheme 3). The (-)-â-elemene (9)
produced in each rearrangement was identified by direct
comparison with an authentic (-)-â-elemene standard by chiral
capillary GC-MS.18 All of the previously described single H309
mutants were also shown to produce (+)-germacrene (7).
Aspartate-Rich Domain. To explore the role of the aspartate-
rich domain of pentalenene synthase, each of the three aspartate
residues was separately replaced by a glutamate. The D80E
mutant suffered a 150-fold decrease in kcat as well as a more
than 20-fold increase in Km, resulting in a net decrease in kcat/
5
measured a reduction in kcat of as much as a factor of 10 .
To probe further the structural basis for the loss of activity
associated with replacement of N219, and to verify that the loss
of activity of the N219L mutant was not due to loss of structural
integrity of the pentalenene synthase active site, we solved the
crystal structure of the N219L mutant and compared it to that
of the wild-type pentalenene synthase. Attempts to also obtain
crystals of the N219L mutant with bound FPP were unsuccess-
ful. The electron density map of N219L pentalenene synthase,
shown in Figure 2A, reveals clear electron density for the
substituted leucine side chain. Moreover, the superposition of
N219L pentalenene synthase with wild-type pentalenene syn-
thase, illustrated in Figure 2B, reveals that the nominally
isosteric amino acid substitution does not result in any significant
conformational changes for neighboring residues in the enzyme
active site, indicating that the active site contour appears to be
conserved in the mutant despite the replacement of the neutral
but polar side chain of N219 with the hydrophobic leucine
3
Km of ∼3.5 × 10 (Table 1). Interestingly, despite the substantial
reduction in catalytic efficiency compared to the efficiency of
the wild-type synthase, the sole product generated by the D80E
mutant was pentalenene. By contrast, the D81E mutant, which
exhibited a 400-fold reduction in kcat/Km compared to the wild
type’s value, due to a combination of a nearly 50-fold decrease
in kcat and a 9-fold increase in Km, generated a mixture of 92%
pentalenene and 4.5% germacrene A (7), accompanied by an
additional 3.5% of an as-yet unidentified sesquiterpene hydro-
carbon, m/z 204. Finally, the D84E mutant, which displayed
relative minor changes in steady-state parameters (2.5-fold
increase in measured kcat offset by a 7-fold increase in Km),
generated a 97:3 mixture of pentalenene and 7.
8a
residue.
Asparagine-219. We have previously noted that the side
chain of asparagine-219 protrudes into the active site cavity of
Overall, the final refined structure of N219L pentalenene
synthase at 2.9 Å is very similar to that of the wild-type enzyme
8a
pentalenene synthase (Figure 1A). Originally, we had proposed
that this residue might influence the cyclization reaction, by
(Figures 2B and 3). There are, however, some minor but
interesting differences in the upper active site region when the
two structures are compared. In the wild-type pentalenene
synthase structure, the F158-D164 polypeptide loop connecting
helices F and G at the rim of the active site is disordered in
(
17) Takeda, K. Tetrahedron 1974, 30, 1525-1534. Weinheimer, A. J.;
Youngblood, W. W.; Washecheck, P. H.; Karns, T. K. B.; Ciereszko, L.
S. Tetrahedron Lett. 1970, 7, 497-500.
(
18) K o¨ nig, W. A.; Rieck, A.; Hardt, I.; Gehrcke, B.; Kubeczka, K.-H.; Muhle,
H. J. High Resolut. Chromatogr. 1994, 17, 315-320. De Kraker, J.-W.;
Franssen, M. C. R.; de Groot, A.; K o¨ nig, W. A.; Bouwmeester, H. J. Plant
Physiol. 1998, 117, 1381-1392.
8a
both monomers, A and B, of the asymmetric unit. In monomer
A of the N219L mutant, this polypeptide loop is similarly
7684 J. AM. CHEM. SOC.
9
VOL. 124, NO. 26, 2002

Pentalenene Synthase
A R T I C L E S
Scheme 4
phenylalanines-76 and -77, are found on helix D, immediately
below the aspartate-rich domain, with the faces of the phenyl
rings angled toward the active site in a sort of open-faced
sandwich (Figure 1A). Although both the F76A and F77A
mutants were constructed, the resultant proteins were obtained
in low yield and primarily as insoluble inclusion bodies, even
when lower temperatures and reduced concentrations of IPTG
were used for induction of protein expression. On the basis of
experiments with partially purified soluble components of each
mutant extract, the activity of each mutant was estimated as no
more than 10% that of the wild-type enzyme, and probably
considerably less. Attempted solubilization and refolding of the
F76A mutant gave only inactive protein. On the other hand,
the F77Y mutant was readily expressed and purified in soluble
form. The kcat and the kcat/Km of the F77Y mutant were 20-fold
lower than those of the wild-type pentalenene synthase (Table
Figure 3. Least-squares ribbon-plot superposition of N219L pentalenene
synthase (green) and wild-type pentalenene synthase (blue) generated with
4
0
41
MOLSCRIPT and Raster3D. Helices F, G (partially hidden), H, and I
are indicated; N219 in the wild-type and the substitute amino acid side
chain, L219, are shown on helix H. The aspartate-rich region is red in both
structures.
disordered, while in monomer B, the corresponding polypeptide
loop is fully ordered in the electron density map. As a
consequence, segmental shifts of R-helical segments in the
region of this loop are evident in the superposition of monomers
B in the wild-type pentalenene synthase and N219L mutant
structures: 1-2 Å segmental shifts are greatest for the upper
part of helices G and H (starting at the site of the N219L
substitution on helix H) and for all of helix I (Figure 3).
Although it is likely that these structural changes occur in
monomer B as a consequence of the N219L substitution on helix
H, it is not clear why these structural changes are observed only
in monomer B of the asymmetric unit and not in monomer A
of the mutant. Neither the wild-type protein nor the N219L
1
). The latter mutant produced predominantly pentalenene (2),
along with very minor amounts of as-yet unidentified sesqui-
terpene hydrocarbons, none of which corresponded to either
germacrene A or protoilludene.
Discussion
Active Site Base. In the crystal structure of pentalenene
synthase, the only apparent basic residue in the active site cavity
8
a
is histidine-309 (Figure 1A). This observation originally led
us to suggest that the imidazole side chain of H309 might serve
as the functional active site base, an inference which was firmly
excluded, however, when site-directed mutagenesis showed that
1
6
His309 is not required for catalytic activity. Although the
H309A, H309C, H309S, and H309F mutants all retained
substantial pentalenene synthase activity, each of these mutants
was found to produce up to 20% of varying proportions of two
sesquiterpene hydrocarbons identified as (+)-germacrene A (7)
and protoilludene (8). The formation of protoilludene (8) by
each of these mutants is readily explained by direct cyclization
of the natural seco-illudyl cation intermediate 5 followed by
deprotonation of the resulting tricyclic protoilludyl cation (12)
2+
mutant shows any bound Mg , similar to free aristolochene
8
c
8b
synthase and trichodiene synthase, but in contrast to epi-
aristolochene synthase.8d
The majority of plant sesquiterpene and monoterpene syn-
thases have an aspartate instead of an asparagine in the
2
+
conserved Mg -binding triad at the position corresponding to
N219 of pentalenene synthase. Reasoning that some or all of
the original cyclase activity might be retained when asparagine
was replaced with aspartate, we also prepared the N219D
mutant. The latter variant indeed showed pentalenene synthase
activity, albeit at a lower level than wild-type, with a 60-fold
decrease in kcat and a 55-fold increase in the Km for FPP,
resulting in a ca. 3300-fold decrease in kcat/Km. Intriguingly,
the N219D mutant produced 9% of a new hydrocarbon which
was shown to be identical by GC-MS with an authentic sample
of â-caryophyllene (10) (Scheme 4). Neither of the previously
observed derailment products, 7 or 8, were observed in
incubations with N219D. The formation of â-caryophyllene (10)
is readily explained by an alternative cyclization of the normal
humulyl cation intermediate 3 and removal of a proton from
the methyl group of the resulting bicyclic cation 11.
(Scheme 2). Protoilludene (8) has previously been isolated from
cultures of Fomitopsis insularis that produce the structurally
1
9
related sesquiterpene fomannosin. Formation of germacrene
A (7) requires an anomalous Markovnikov cyclization of the
originally generated allylic cation at C-10 of the 10,11-double
bond of FPP, followed by deprotonation of one of the two
attached methyl groups (Scheme 2). Germacrene A (7) is itself
a common sesquiterpene metabolite that has also been implicated
as an intermediate in the formation of a wide variety of bicyclic
eudesmane, eremophilane, and vetispirane sesquiterpenes, in-
1,18,20
cluding both aristolochene and epi-aristolochene.
Notably,
the formation of the (+)-enantiomer of 7 is consistent with the
Aromatic Residues. Among the many aromatic residues
which line the active site of pentalenene synthase, two of these,
(
19) Furukawa, J.; Morisaki, N.; Kobayashi, H.; Iwasaki, S.; Nozoe, S.; Okuda,
S. Chem. Pharm. Bull. 1985, 33, 440-443.
J. AM. CHEM. SOC.
9
VOL. 124, NO. 26, 2002 7685

A R T I C L E S
Seemann et al.
well-documented absolute sense of folding of FPP in the
formation of pentalenene. Both anomalous cyclization products,
(3) Finally, the active site base might be the tightly bound
2
3
pyrophosphate counterion. It is not immediately clear, how-
ever, that this group would be capable of reaching all of the
various sites of deprotonation that lead to the formation of
pentalenene and its three identified derailment products. Resolu-
tion of these issues will require high-resolution crystal structures
of protein bound to substrate or intermediate analogues.
Binding of the Divalent Cation. The aspartate-rich domain,
which is universally conserved in all terpene synthases, has been
shown to play an important role in binding of the requisite
(+)-germacrene A (7) and protoilludene (8), result from
derailment of the normal cyclization cascade, presumably due
to relaxed control over the conformation of the substrate FPP
and derived intermediates in the active sites of the various H309
mutants.
In their analysis of the structure of epi-aristolochene synthase,
Chappell and Noel had postulated that an active site tryptophan
(W273) might serve as a Lewis base, reasoning that the indole
2
+ 1,2,10,13,14
ring is intrinsically reactive toward acid and other electrophilic
divalent cation, which is usually Mg .
Crystal struc-
8
d,21
reagents.
In support of this suggestion, they reported that
tures of both epi-aristolochene synthase with a bound FPP
8
d
replacement of W273 by either aromatic or nonaromatic amino
acid residues abolished cyclase activity in the epi-aristolochene
synthase. Since pentalenene synthase has a tryptophan residue
analogue, as well as of trichodiene synthase with bound
8
b
inorganic pyrophosphate, have revealed that, in fact, three
2
+
Mg ions are bound at the active site, two of which are
liganded, either directly or indirectly, by two or more of the
carboxylates of the DDXXD motif. These two Mg2 ions in
turn form a complex with the pyrophosphate moiety of the
substrate. Together with a third magnesium ion, which is held
on the opposite side of the pyrophosphate group by the (N/D)-
DXX(S/T)XXXE triad of amino acid side chains, the divalent
metal ions serve to bind and orient the pyrophosphate group of
the substrate FPP, while simultaneously activating the allylic
diphosphate moiety for the ionization that initiates the cascade
of electrophilic cyclization reactions. Consistent with this model,
replacement of the individual aspartate residues in each of two
aspartate-rich domains in the active site of farnesyl diphosphate
synthase resulted in nearly complete loss of prenyl transferase
(W308) immediately adjacent to the previously examined
+
His309, we generated two mutant pentalenene synthases in
which this tryptophan was replaced by phenylalanine, W308F,
and the double mutant, W308F/H308F (Table 1). Both mutants
retained substantial pentalenene synthase activity. Although
neither mutant produced detectable protoilludene (8), the W308F
and W308F/H309F mutants also generated varying proportions
of (+)-germacrene A (7). Together these experiments firmly
exclude either histidine-309 or tryptophan-308 as the active site
base for pentalenene synthase.
Deprotonation of the carbocationic intermediates of terpenoid
cyclization does not require an especially potent base, since
tertiary carbocations are highly acidic, with estimated pKa values
in the range of -10.22 The stereospecific removal of H-7re of
24
activity. By contrast, the corresponding D100E, D101E, and
6
that generates pentalenene (2) is certainly not a spontaneous
D104E mutants of trichodiene synthase display only modest
changes in steady-state kinetic parameters (but up to 100-fold
decreases in the slowest chemical step, that of FPP ionization),
while generating a mixture of five anomalous sesquiterpene
cyclization products in addition to the normal cyclization
chemical event and is presumably mediated by a discrete,
dedicated active site base. It is geometrically unlikely, however,
that this same base can also mediate each of the anomalous
deprotonations that generate the various derailment products
such as germacrene A, protoilludene, and â-caryophyllene. Since
we have ruled out the only nominally basic residues present in
the active site, His309 and the adjacent Trp308, as active site
bases, how can one now account for the action of apparently
multiple bases? At least three possibilities must be considered:
1
4
product, trichodiene. Recent crystallographic studies of the
D100E mutant of trichodiene synthase with bound inorganic
pyrophosphate have revealed that the complex coordinates only
2
+
two of the three Mg ions and that the volume of the active
3
site cavity has increased some 12%, from 324 to 364 Å ,
(
1) The base or bases could be enzyme-bound water(s), given
allowing for greater spatial and conformational degrees of
freedom in the binding of the substrate and derived inter-
+
22
that the pKa of the conjugate acid H3O is -2 to -4.
A
8
b
potential drawback to water acting as the active site base,
however, is its intrinsic nucleophilicity, which could lead to
the competing formation of alcohol side products, none of which
are observed for either the wild-type cyclase or any of the
mutants examined. In principle, this nucleophilicity could be
suppressed if the bound water were to be tightly constrained
within the active site. So far, there is no crystallographic
evidence for or against the presence of water bound at the active
site in the presence of substrates or analogues. (2) The carbonyl
oxygen atoms of the peptide backbone are also basic enough
mediates. For pentalenene synthase, the D80E, D81E, and
D84E mutants also show an analogous degradation in catalytic
power and product specificity, although there is no simple
correlation between the magnitude of the change in steady-state
kinetic parameters and the relative proportions of abortive
cyclization products. Since release of the final sesquiterpene
products is likely to be rate-limiting, as has been demonstrated
for both trichodiene synthase and epi-aristolochene synthase,25
even a 10-fold decrease in the rate of the slowest chemical step
in the pentalenene synthase reaction sequence could well be
masked by an even slower product-off step.
(
pKa of protonated amide ) 0 to -2)22 to accept a proton from
the C-7 methylene group of 6 and other cationic intermediates.
Asparagine-219 of pentalenene synthase is part of a highly
conserved motif, (L,V)(I,V,L)(N,D)D(I,L,V)A(S,T)XXXE, that
(
(
(
20) Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc.
2
1
000, 122, 1861-1866. Cane, D. E.; Tsantrizos, Y. S. J. Am. Chem. Soc.
996, 118, 10037-10040.
21) Biswas, K. M.; Jackson, A. H. J. Chem. Soc., Perkin Trans. 1 1989, 11,
(23) Davisson, V. J.; Neal, T. R.; Poulter, C. D. J. Am. Chem. Soc. 1993, 115,
1235-1245.
1
981-1986. Abdullah, M. I.; Jackson, A. H.; Lynch, P. P.; Record, K. A.
(24) Marrero, P. F.; Poulter, C. D.; Edwards, P. A. J. Biol. Chem. 1992, 267,
21873-21878. Song, L. S.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 3044-3048.
F. Heterocycles 1990, 30, 317-320.
22) Deno, N. C. Prog. Phys. Org. Chem. 1964, 2, 129-193. Lowry, T. H.;
Richardson, K. S. Mechanism and Theory in Organic Chemistry; Harper
(25) (a) Cane, D. E.; Chiu, H.-T.; Liang, P.-H.; Anderson, K. S. Biochemistry
1997, 36, 8332-8339. (b) Mathis, J. R.; Back, K.; Starks, C.; Noel, J.;
Poulter, C. D.; Chappell, J. Biochemistry 1997, 36, 8340-8348.
&
Row: New York, 1976; pp 149-151. See also http://webbook.nist.gov/
chemistry/
7686 J. AM. CHEM. SOC.
9
VOL. 124, NO. 26, 2002

Pentalenene Synthase
A R T I C L E S
is found in all monoterpene, sesquiterpene, and diterpene
synthases.15 Both N225 of trichodiene synthase and D445 of
Scheme 5
8b
8d
epi-aristolochene synthase, each of which is known to be
chelated to a Mg2 ion, are found at nearly identical positions
within the active sites of their respective enzymes as N219 and
N244 of pentalenene and aristolochene synthase, and all four
residues have their side chain carbonyl groups oriented in the
same manner. Consistent with the expected role of N219 in
+
2+
binding Mg , the corresponding N219A and N219L mutants
of pentalenene synthase are both completely inactive. Analysis
of the crystal structure of the latter mutant reveals no significant
changes in protein structure or in the shape of the active site
bond and deprotonation of the resulting tertiary cation 13
(Scheme 5, pathways b and c). Is formation and trapping of the
germacradienyl cation 13 an anomalous process that occurs in
competition with direct formation of the humulyl cation 3 only
in pentalenene synthase mutants in which the substrate is
anomalously bound, or is the germacradienyl cation 13 a natural,
but normally cryptic, intermediate of the wild-type cyclization
reaction, that ordinarily undergoes rapid Wagner-Meerwein
rearrangement (Scheme 5, pathway d) to the secondary humulyl
cation 3?
(
Figures 2 and 3). Notably, we have also recently found that
the N244L mutant of aristolochene synthase is inactive, as
2
6
predicted. Croteau has recently reported that the homologous
N765A mutant of abietadiene synthase, a diterpene synthase
originally isolated from grand fir (Abies grandis), suffers a more
than 20 000-fold decrease in kcat compared to the value for the
wild type, while substitution of alanine for either T769 or E773,
the other two components of the Mg -binding triad, reduced
kcat by 10 000-fold and 5000-fold, respectively.27 On the other
hand, replacement of N219 of pentalenene synthase with
aspartate, the amino acid that is found at the homologous
position of the majority of plant monoterpene, sesquiterpene,
and diterpene synthases, restores pentalenene synthase activity
to ca. 0.03% of its wild-type level, while resulting in concomi-
tant formation of the anomalous side-product â-caryophyllene
2+
The apparent selective formation of the nominally less stable,
anti-Markovnikov addition product is, in fact, a very common
theme in the biosynthesis of cyclic terpenoids, from monoter-
1,2,28
penes to triterpenes.
This apparent divergence from ordinary
solution or gas-phase kinetic behavior has motivated a variety
of proposals to account for the apparently divergent chemistry
of enzyme-catalyzed terpene synthase reactions. For example,
on the basis of extensive studies of the cyclization of hetero-
atom-substituted squalene analogues by lanosterol synthase,
Corey and others have argued that, in the generation of ring C
of steroids and other triterpenes, the apparent direct, anti-
Markovnikov cyclization of the bicyclic intermediate 14 leading
to the formation of the tricyclic secondary cation 15 (Figure
4A, pathway a) can be avoided if one posits an initial
Markovnikov cyclization of 14 to generate the tertiary cyclo-
(10) (Scheme 4).
Carbocation Stabilization. Both theoretical and experimental
studies have provided support for the importance of aromatic
amino acid residues in stabilizing carbocations at enzyme active
sites.12 Among the many aromatic residues that can be seen
within the active site of pentalenene synthase, F76 and F77
appear to be well placed so as to interact with positive charge
at C-1, C-2, and C-3 of the farnesyl residue and derived
intermediates (Figure 1A). Replacement of either F76 or F77
by alanine led to at least a 10-fold reduction in catalytic activity,
but the low yield and poor solubility of the mutant proteins
precluded more quantitative analysis of the steady-state kinetic
parameters. Mutant F77Y, in which the phenylalanine is replaced
by a more electron-rich tyrosine residue, produced predomi-
nantly pentalenene, with essentially no change in Km compared
to the value for the wild type but with kcat reduced by a factor
of 20, suggesting that the precise geometry of interaction of
the aromatic residues with the intermediate carbocations is
critical to enhancement of the rate of the reaction.
The currently accepted mechanism for the pentalenene
synthase reaction involves ionization of the substrate FPP and
electrophilic attack of C-1 of the initially generated allylic cation
at C-11 of the 10,11-double bond of FPP to give the secondary
humulyl cation 3 (Scheme 5, pathway a). This anti-Markovnikov
regiochemistry contrasts with solution-phase reactions in which
electrophilic additions normally occur at the less substituted end
of a double bond to give the more highly substituted, thermo-
dynamically more stable carbocation. In those mutants of
pentalenene synthase in which (+)-germacrene A (7) is gener-
ated in addition to pentalenene, formation of 7 results from
competing Markovnikov addition to C-10 of the 10,11-double
2
9
pentylcarbinyl cation 16 (Figure 4A, pathway b). The latter
intermediate would then undergo ring expansion to generate the
secondary cyclohexyl cation 15 (Figure 4A, pathway c). Detailed
calculations carried out by Jorgensen, using both ab initio and
molecular mechanics methods to model the cyclization-
rearrangement in a condensed phase designed to mimic the
environment of aromatic residues thought to be at the active
site of lanosterol synthase, predicted that direct Markovnikov
cyclization of 14 to 16 would be exothermic by > -8 kcal/
mol, while subsequent rearrangement of 16 to the secondary
30
cation 15 would be endothermic by 12 kcal/mol. These results
were interpreted as being consistent with the cyclization-
rearrangement pathway proposed by Corey. On the other hand,
since the free energy of the ultimately formed secondary cation
15 cannot depend on the pathway by which it is formed,
generation of 15 from the hypothetical Markovnikov intermedi-
ate 16 must be at least 8 kcal/mol more endothermic than direct
(
28) Abe, I.; Prestwich, G. D. In Isoprenoids Including Carotenoids and Steroids;
Cane, D. E., Ed. (Volume 2 in ComprehensiVe Natural Products Chemistry;
Barton, D, Nakanishi, K., Meth-Cohn, O., Eds.); Pergamon Press: Elsevi-
er: Oxford, 1999; pp 267-298. Poralla, K. In Isoprenoids Including
Carotenoids and Steroids; Cane, D. E., Ed. (Volume 2 in ComprehensiVe
Natural Products Chemistry; Barton, D, Nakanishi, K., Meth-Cohn, O.,
Eds.); Pergamon Press: Elsevier: Oxford, 1999; pp 299-319.
(29) Corey, E. J.; Virgil, S. C.; Cheng, H. M.; Baker, C. H.; Matsuda, S. P. T.;
Singh, V.; Sarshar, S. J. Am. Chem. Soc. 1995, 117, 11819-11820. Wendt,
K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R. Angew. Chem., Int. Ed. 2000,
39, 2812-2833.
(
26) Felicetti, B.; Cane, D. E. Unpublished observations.
(
27) Peters, R. J.; Croteau, R. B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 580-
5
84.
(30) Jenson, C.; Jorgensen, W. L. J. Am. Chem. Soc. 1997, 119, 10846-10854.
J. AM. CHEM. SOC.
9
VOL. 124, NO. 26, 2002 7687

A R T I C L E S
Seemann et al.
Figure 4. (A) Markovnikov (b) versus anti-Markovnikov (a) cyclization of the lanosterol precursor 14. (B) Competing Markovnikov (a) and anti-Markovnikov
b) cyclization of the R-terpinyl cation 18 in the formation of R-pinene, â-pinene, and camphene by pinene synthase.
(
generation of 15 from 14 by the supposedly forbidden anti-
Markovnikov process. In fact, the calculations themselves show
the tricyclic secondary cation 15 to be no more than 4 kcal/mol
higher in free energy than the bicyclic precursor 14, most of
the driving force for the reaction coming from the net conversion
of a double bound to a single bond. There is thus no direct
experimental evidence or theoretical support for an intrinsic
thermodynamic (or, more importantly, kinetic) advantage to the
proposed two-step cyclization-rearrangement process; indeed,
such a pathway would require a thermodynamically unfaVorable
rearrangement step after the hypothetical Markovnikov cycliza-
tion.
synthase-catalyzed cyclization of FPP, formation of the second-
ary humulyl cation 3 is therefore expected to take place directly,
without intervention of the otherwise more stable germacradienyl
cation 13 (Scheme 5). Formation of (+)-germacrene A (7) by
the various pentalenene synthase mutants is thus a true derail-
ment or diversion of the normal cyclization reaction, and not
simply the consequence of trapping of a normally cryptic,
cationic intermediate.
Experimental Section
Materials. Sources of reagents and chromatographic materials were
as previously described.6 Expression, purification, and assay of
recombinant pentalenene synthase from E. coli BL21(DE3)/pZW05 was
6
To address the issue of competing formation of Markovnikov
and anti-Markovnikov products during enzyme-catalyzed ter-
penoid biosynthesis, we have previously used the powerful
perfomed as described. Authentic â-caryophyllene was obtained from
Sigma. Mutagenic oligonucleotides and primers were purchased from
IDT. The Altered Sites II in vitro Mutagenesis System and competent
cells of ES1301mutS and JM109 were obtained from Promega, while
E. coli BL21(DE3) was purchased from Stratagene. EcoRI, HindIII,
and T4 DNA ligase were purchased from Stratagene, and AgeI, PpuMI,
SacI, and EcoNI were from New England Biolabs. Plasmid DNA
purification was performed using the QIAprep spin Miniprep kit
31
technique of isotopically sensitive branching. (-)-Pinene
synthase is a monoterpene synthase which converts geranyl
diphosphate to a mixture of several monoterpenes, including
(
-)-R-pinene, (-)-â-pinene, and (-)-camphene.32 Cyclization
of a deuterated sample of GPP resulted in an increase in the
ratio of R-pinene to â-pinene, due to the isotope effect on
removal of deuterium from the methyl group of the common
bicyclic intermediate pinanyl cation 17, without perturbing the
ratio of total R- and â-pinenes to camphene31 (Figure 4B). These
(
Qiagen).
General Methods. Restriction endonuclease digestions, DNA liga-
tions, transformation of competent cells, plasmid minipreps, mutagen-
esis, and other standard recombinant DNA manipulations were carried
out by standard methods and according to the suppliers’ protocols.
dsDNA sequencing was performed by the HHMI Biopolymer/Keck
Foundation Biotechnology Resource Laboratory at the Yale University
School of Medicine, New Haven, CT, using the dideoxy dye terminator
method and automated fluorescence sequencing. Analysis of DNA and
protein sequences as well as PCR primer design utilized the suite of
programs in the Wisconsin sequence analysis package, version 10.0
results established that the commitment to formation of cam-
phene must take place before the isotopically sensitive step and
that cyclization of the R-terpinyl cation 18 therefore involves
competing Markovnikov and anti-Markovnikov cyclizations.
Had only Markovnikov cyclization of the R-terpinyl cation been
allowed, camphene would have had to be formed by rearrange-
ment of the common pinanyl cation intermediate 17 to the bornyl
cation 19 (Figure 4B, pathway c), rather than by direct formation
of 19 from the R-terpinyl cation (Figure 4B, pathway b). In
such a case, the deuterium isotope effect would have increased
the proportion of both camphene and R-pinene relative to
â-pinene. To our knowledge, the isotopically sensitive branching
experiment is by far the clearest and least ambiguous demon-
stration that there is no intrinsic prohibition against enzyme-
catalyzed anti-Markovnikov reactions. Such “disfavored” elec-
trophilic additions are presumably facilitated by specific enzymic
stabilization of the nominally less stable transition state leading
to the secondary carbocationic intermediate. In the pentalenene
(Unix), Genetics Computer Group (GCG), Madison, WI.
Pentalenene Synthase Mutants. The EcoRI-HindIII fragment of
6
pZW05 which harbors the wild-type pentalenene synthase gene was
ligated into the Promega vector pALTER-1, and mutations were
introduced using the Altered Sites II in vitro Mutagenesis system in
combination with the mutagenic primers listed in Table S1 of the
Supporting Information. Mutant plasmids were initially screened by
restriction digestion to verify the loss or introduction of any relevant
restriction sites (Table S1, Supporting Information). The identities of
each of the mutations were then directly verified by sequencing of the
full-length gene. Each of the mutant genes was then excised by EcoRI-
HindIII digestion, ligated back into the T7-based expression plasmid
pLM1, and subcloned in E. coli XL1 Blue. The resultant mutant
plasmids were then used to transform the expression host E. coli BL21-
(
DE3).
Protein Overexpression and Kinetic Studies. Individual strains
of E. coli BL21(DE3) harboring the mutant plasmids pGZ11 (H309A),
(
31) Croteau, R. B.; Wheeler, C. J.; Cane, D. E.; Ebert, R.; Ha, H.-J. Biochemistry
987, 26, 5383-5389.
32) Gambliel, H.; Croteau, R. J. Biol. Chem. 1984, 259, 740-748.
1
(
7
688 J. AM. CHEM. SOC. VOL. 124, NO. 26, 2002
9

Pentalenene Synthase
A R T I C L E S
pGZ12 (H309S), pGZ13 (H309C), pMS11 (H309F), pMS04 (W308F),
pMS03 (W308F/H309F), pMS05 (D80E), pMS06 (D81E), pMS08
mm plate, crystal-to-detector distance ) 120 mm, λ ) 1.08 Å). Crystals
of N219L pentalenene synthase were isomorphous with those of the
wild-type enzyme.8a A total of 63 768 measured reflections yielded
(D84E), pGZ14 (N219A), pGZ17 (N219L), pMS07 (N219D), and
pMS09 (F77Y) were each grown overnight in 50 mL of LB media
23 110 unique reflections, 97% complete to 2.9 Å resolution (Rsym )
containing 100 µg/mL ampicillin at 37 °C and 250 rpm. This culture
0.083). Data integration and reduction was performed using the HKL
(10 mL) was used to inoculate 500 mL of the same media which was
program suite,33 and corrected structure factor amplitudes were gener-
3
4
grown for 3 h until OD600 > 0.8. The culture was transferred to a bath
at 30 °C, and after 30 min enzyme production was induced using 0.5
mM IPTG for 3 h. After centrifugation (6000g, 25 min), 2.8 g of cells
was typically obtained. Lysis of the cells, DNAse treatment, and
ated with routines contained in the CCP4 program suite.
Initial phases for the electron density map of N219L pentalenene
synthase were obtained by molecular replacement against 20-4 Å data
using routines contained in CNS35 and residues 9-304 of the wild-
6
8a
sonication were performed as for wild-type pentalenene synthase. After
type pentalenene synthase crystallization dimer as a search probe. The
PEG precipitation of the undesired proteins and centrifugation (9500g,
cross-rotation function and subsequent translation searches yielded an
unambiguous solution with a correlation coefficient significantly higher
than those for all other solutions (0.436 with the next closest peak at
0.197): R ) 114.69°, â ) 175.81°, γ ) 52.28°, x ) 0.83, y ) -3.60,
z ) -1.88. Rigid-body refinement lowered the initial R factor from
0.455 to 0.432, and subsequent refinement employed torsion angle
dynamics with energy minimization (Tinitial ) 5000 K), using the
2
0 min), the mutant pentalenene synthase was purified as previously
6
described. Kinetic assays were performed as previously described for
wild-type pentalenene synthase, except that different ranges of con-
centration for [1- H]FPP were used to compensate for the variations
3
m
in K . Steady-state kinetic parameters were determined by direct fitting
of the data to the Michaelis-Menten equation by nonlinear least-squares
regression using the commercial Kaleidagraph software package.
Mutant Product Analysis. In a typical incubation reaction, 100 µg
of purified enzyme was incubated with FPP (50 µM) in a total volume
of 1 mL supplemented with kinetics buffer (50 mM Tris-HCl, 5 mM
35
maximum likelihood algorithm implemented in CNS. Model building
was performed using O.36 Strict noncrystallographic symmetry con-
straints and grouped B factors (one main chain and one side chain per
residue) were initially imposed in refinement. However, improved
convergence was achieved when a bulk solvent correction was applied,
when restrained individual B factors were utilized, and when noncrys-
tallographic symmetry constraints were converted to highly weighted
2
MgCl , 1 mM DTT, 0.2 mM PMSF, 0.2 mM benzamidine, 20% (v/v)
glycerol, pH 8.2) at 30 °C for 1 h, and the aqueous layer was overlaid
with 2 mL of HPLC grade pentane. The hydrocarbon products were
extracted twice with 1 mL of pentane. The organic layer was
concentrated to about 100 µL under reduced pressure at 0 °C. The
concentrate was analyzed by GC-MS using a HP-5988A GC-MS
system containing a HP-1 column and running the following heating
program: 55 °C for 4 min and then 5 °C/min until 210 °C with an
injector temperature of 150 °C. Under those conditions, pentalenene
-
1
restraints (w ) 100-300 kcal mol ) in loop regions. Residues 7-311
were fit into the final electron density map along with a total of 26
water molecules.
Refinement converged smoothly to a final crystallographic R factor
of 0.263 (Rfree ) 0.308). The final model had excellent stereochemis-
try: root-mean-square (rms) deviations from ideal bond lengths and
angles were 0.009 Å and 1.3°, respectively, and analysis with
PROCHECK37 indicated 464, 48, and 2 nonproline/nonglycine residues
in both molecules of the asymmetric unit with most favored, additionally
allowed, and generously allowed backbone conformations, respectively.
Refined atomic coordinates of N219L pentalenene synthase have been
deposited in the Research Collaboratory for Structural Bioinformatics
with PDB accession code 1HM7.
(2), (+)-germacrene A (7), protoilludene (8), and â-caryophyllene (9)
eluted at 15.53, 19.33, 16.45, and 17.30 min, respectively.
Determination of the Stereochemistry of Germacrene A. The
germacrene samples from the H309F, H309S, H309C, W308F, and
W308F/H309F mutants were each shown to correspond to (+)-
germacrene A (7). The absolute configuration of (+)-germacrene A
was demonstrated by means of its Cope rearrangement to (-)-â-elemene
1
7
(
9). The sample in hexane (2 µL) was analyzed by GC-MS (25 m
0.25 mm i.d. heptakis(6-O-TBDMS-2,3-di-O-methyl)-â-cyclodextrin
50% in OV17)) using an injection port temperature of 250 °C to induce
Acknowledgment. This research was supported by an NIH
Merit Award (GM30301) to D.E.C. and an NIH Grant
(GM56838) to D.W.C. We also thank F. Verstappen for
technical assistance and Prof. W. A. K o¨ nig of Hamburg
University, Germany, for the gift of the â-elemene standards.
×
(
the rearrangement of enzymatically produced (+)-germacrene A (7),
with an oven temperature program of 4 min at 45 °C followed by a
-
1
ramp of 2 °C min to 170 °C. Spectra were recorded in the selected
ion monitoring mode (m/z 121, 147, and 189). A racemic â-elemene
standard was isolated from the hydrodistillate of the liverwort Frullania
macrocephalum, and the elution order of its enantiomers was determined
Supporting Information Available: Table S1 listing mu-
tagenic primers for generation of pentalenene synthase mutants,
and GC-MS chromatograms from analysis of enzymatic
formation of (+)-germacrene (7) (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
18
with a (-)-â-elemene standard. Under these conditions, (+)-â-elemene
and (-)-â-elemene eluted at 31.82 and 31.97 min, respectively.
Crystallography. Purified N219L pentalenene synthase was con-
centrated to 2-7 mg/mL using Centricon filter devices (Amicon) and
used in sitting-drop batch crystallization trials at room temperature:
various protein concentrations [2.0, 4.5, and 6.6 mg/mL N219L
pentalenene synthase in 100 mM HEPES (pH ) 7.0)], protein solution
volumes, and precipitant buffer volumes were sampled in 3 × 3 Pyrex
JA026058Q
(
33) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326.
34) Collaborative Computational Project, Number 4. Acta Crystallogr. 1994,
D50, 760-763.
(
(
35) Br u¨ nger, A. T.; Adams, P. D.; Clore, G. M.; Gros, P.; Grosse-Kunstleve,
R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, N.; Pannu, N. S.; Read, R. J.;
Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr. 1998, D54,
905-921.
(Corning) grids sealed in plastic containers and equilibrated against
3
5 mL of precipitant buffer [1.5 M ammonium sulfate, 100 mM Tris-
HCl (pH ) 8.2)]. The optimal composition of sitting drops was 40 µL
of enzyme solution and 20 µL of precipitant buffer, which yielded
hexagonal, rodlike crystals of approximate dimensions 0.2 mm × 0.2
mm × 0.4 mm within 3 weeks. These crystals were prepared for X-ray
data collection by gradual transfer into a 30% glycerol cryoprotectant
solution and flash-cooled in liquid propane for transport to the Stanford
Synchrotron Radiation Laboratory (Beamline 7-1). X-ray diffraction
data were collected using a MAR 345/CCD image plate system (300
(36) Jones, T. A.; Zou, J.-Y.; Cowan, S. W.; Kjeldgaard, M. Acta Crystallogr.
1991, A47, 110-119.
(37) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J.
Appl. Crystallogr. 1993, 26, 283-291.
(
38) Swiss-Pdb Viewer (Deep View): http://www.expasy.ch/spdbv/mainpage-
.htm
(39) Nicholls, A.; Honig, B. J. Comput. Chem. 1991, 12, 435-445.
(
(
40) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946-950.
41) Bacon, D. J.; Anderson, W. F. J. Mol. Graph. 1988, 6, 219-220. Merritt,
E. A.; Murphy, M. E. P. Acta Crystallogr. 1994, D50, 869-873.
J. AM. CHEM. SOC.
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