Structure of epi-isozizaene synthase from streptomyces coelicolor A3(2), a platform for new terpenoid cyclization templates

Article abstract of DOI:10.1021/bi902088z

The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 A resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg²⁺ ions, inorganic pyrophosphate (PP_i), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 A resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation π interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 A resolution crystal structure of F198A EIZS in a complex with three Mg²⁺ ions, PP_i, and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg ²⁺ ions has been determined at 1.90 A resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

Full text of DOI:10.1021/bi902088z

Biochemistry 2010, 49, 1787–1797 1787
DOI: 10.1021/bi902088z
Structure of Epi-Isozizaene Synthase from Streptomyces coelicolor A3(2), a Platform for
New Terpenoid Cyclization Templates†,‡
Julie A. Aaron,§ Xin Lin, David E. Cane,*, and David W. Christianson*,§
§Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania
19104-6323, and Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108
Received December 5, 2009; Revised Manuscript Received January 20, 2010
ABSTRACT: The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase
˚
from Streptomyces coelicolor A3(2), has been determined at 1.60 A resolution. Specifically, the structure of
wild-type EIZS is that of its closed conformation in complex with three Mg2þ ions, inorganic pyrophosphate
(PPi), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been
˚
determined in an open, ligand-free conformation at 1.90 A resolution. Comparison of these two structures
provides the first view of conformational changes required for substrate binding and catalysis in a bacterial
terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation
intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented
to stabilize carbocation intermediates in the cyclization cascade through cation-π interactions. Mutagenesis
of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product
arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the
˚
cyclization of farnesyl diphosphate. Accordingly, the 1.64 A resolution crystal structure of F198A EIZS in a
complex with three Mg2þ ions, PPi, and BTAC reveals an alternative binding orientation of BTAC;
alternative binding orientations of a carbocation intermediate could lead to the formation of alternative
products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg2þ ions has been
˚
determined at 1.90 A resolution, showing that metal binding triggers a significant conformational change of
helix G to cap the active site.
The terpenome comprises a chemical library of more than
55000 natural products found in all forms of life that serve a
myriad of biological functions. The structural and stereochemical
diversity of these terpenoid metabolites derives in large part from
the action of terpenoid cyclases, which catalyze the carbocation-
mediated cyclization of linear isoprenoids such as the sesquiter-
pene1 farnesyl diphosphate (FPP)2 to generate an enormous
variety of cyclic products (1-5). The active site of a terpenoid
cyclase serves as a template for the binding of the flexible acyclic
isoprenoid substrate in a unique conformation that permits a
specific sequence of bond-making and bond-breaking steps to
result in the ultimate formation of a single cyclic product or, as is
often the case, a mixture of products generated by the more
promiscuous cyclases. Crystal structures of bacterial, fungal, and
plant sesquiterpene cyclasess1 determined to date (6-11) reveal a
common R-helical fold similar to that first observed for avian
FPP synthase (12), despite insignificant amino acid sequence
identity among all of these synthases.
In spite of insignificant overall sequence identity, two metal ion
binding motifs are universally conserved among terpenoid
cyclases: an aspartate-rich motif (DDXXD/E) located at the
C-terminus of helix D and the “NSE/DTE” motif (N/DDXXS/
TXX(K/R)E) at the C-terminus of helix H (bold residues are
usually metal ligands). The electrophilic cyclization cascade
catalyzed by a sesquiterpene cyclase is initiated by the divalent
metal ion-dependent ionization of FPP to generate inorganic
pyrophosphate (PPi) and a highly reactive allylic carbocation that
can undergo rapid isomerization, cyclization, and rearrange-
ment. X-ray crystallographic studies of sesquiterpene cyclases
indicate that significant protein structural changes occur upon
the binding of three Mg2þ ions and PPi or FPP analogues to
trigger active site closure and substrate ionization, but the details
of these structural changes generally differ between plant (7, 11)
and fungal (9, 10) sesquiterpene cyclases. Until now, the mechan-
ism of active site closure of a bacterial terpenoid cyclase has
remained unknown since the only available crystal structure of a
bacterial cyclase has been that of Streptomyces exfoliatus
†Supported by National Institutes of Health (NIH) Grants GM56838
to D.W.C. and GM30301 to D.E.C. and an NIH Chemistry Biology
Interface Training Grant to J.A.A.
‡The atomic coordinates and structure factors of wild-type and
F198A epi-isozizaene synthase in a complex with three Mg2þ ions,
inorganic pyrophosphate, and the benzyltriethylammonium cation;
wild-type epi-isozizaene synthase in a complex with four Hg2þ ions;
and ligand-free D99N epi-isozizaene synthase have been deposited in the
Protein Data Bank as entries 3KB9, 3LG5, 3KBK, and 3LGK,
respectively.
*To whom correspondence should be addressed. D.W.C.: telephone,
(215) 898-5714; fax, (215) 573-2201; e-mail, chris@sas.upenn.edu. D.E.C.:
telephone, (401) 863-3588; fax, (401) 863-9368; e-mail, david_cane@
brown.edu.
1A sesquiterpene is a 15-carbon isoprenoid; farnesyl diphosphate is a
sesquiterpene formed by the linkage of three five-carbon isoprenoids in a
linear, head-to-tail fashion. A sesquiterpene cyclase catalyzes the
cyclization of farnesyl diphosphate to form alternative 15-carbon iso-
prenoid compounds.
2Abbreviations: APS, Advanced Photon Source; BME, β-mercap-
toethanol; BTAC, benzyltriethylammonium cation; EIZS, epi-isozi-
zaene synthase; FPP, farnesyl diphosphate; LB, Luria-Bertani; PDB,
Protein Data Bank; PEG, polyethylene glycol; PPi, inorganic pyropho-
sphate; rmsd, root-mean-square deviation; SAD, single-wavelength
anomalous dispersion; TCEP, tris(2-carboxyethyl)phosphine; WT,
wild-type.
r
2010 American Chemical Society
Published on Web 02/04/2010
pubs.acs.org/Biochemistry

1788 Biochemistry, Vol. 49, No. 8, 2010
Aaron et al.
UC5319 pentalenene synthase, which was determined only in an
open, ligand-free conformation (6).
represent the mutant codon introduced): F96A, 50-CTA CAG
CGC GTG GTT Cgc aGT CTG GGA CGA CCG TC-30 (primer1)
and 50-GAC GGT CGT CCC AGA Ctg cGA ACC ACG CGC
TGT AG-30 (primer 2); D99N, 50-GGT TCT TCG TCT GGa
acG ACC GTC ACG AC-30 (primer 1) and 50-GTC GTG ACG
GTC gtt CCA GAC GAA GAA CC-30 (primer 2); F198A, 50-
GAA CTG CGC CGG CTC ACG gca GCG CAC TGG ATC
TGG AC-30 (primer 1) and 50-GTC CAG ATC CAG TGC GCt
gcC GTG AGC CGG CGC AGT TC-30 (primer 2); W203F,
50-GTT CGC GCA CTG GAT Ctt tAC CGA CCT GCT GG-30
(primer 1) and 50-GCT CCA GCA GGT CGG Taa aGA TCC
AGT GCG CG-30 (primer 2). The optimal reaction mixture for
PCR amplification of the insert was 100 ng of each forward and
reverse primer, 3 μL of 10 mM dNTP mix, 100 ng of plasmid,
5 μL of Pfu turbo polymerase buffer, and 1 unit of Pfu turbo
polymerase diluted with water to a final volume of 50 μL.
Optimal PCR conditions required initial denaturation of the
reaction mixture at 95 ꢀC for 5 min, addition of polymerase
followed by 30 cycles (denaturation for 1 min at 95 ꢀC, annealing
for 1 min at 60 ꢀC, and extension for 8 min at 72 ꢀC), and a final
10 min extension at 72 ꢀC followed by a final hold at 4 ꢀC. One
microliter of Dpn1 was added to the PCR mixture and incubated
at 37 ꢀC for 1 h to digest the template. PCR products were
transformed into XL1-Blue cells for DNA isolation and sequen-
cing. DNA was purified (Qiagen mini-prep kit) from cultures
from single colonies, and DNA sequencing (DNA Sequencing
Facility, University of Pennsylvania) confirmed incorporation of
the mutations. Mutant proteins were expressed and purified
using the same procedures described above for the wild-type
enzyme.
Here, we report the first X-ray crystal structure of a bacterial
cyclase in a closed active site conformation, recombinant epi-
isozizaene synthase (EIZS) from Streptomyces coelicolor A3(2).
EIZS is related to pentalenene synthase by 24% amino acid
sequence identity and catalyzes the multistep cyclization of FPP
to form the tricyclic hydrocarbon epi-isozizaene (13, 14). The
gene for EIZS is itself transcriptionally coupled to that of
cytochrome P450 CYP170A1 (15) which performs two sequential
allylic oxidations on epi-isozizaene to form albaflavenone, an
antibiotic with activity against Bacillus subtilis (16). We have
determined the structure of the EIZS-Mg2þ3-PPi-benzyl-
˚
triethylammonium cation (BTAC) complex at 1.60 A resolution,
˚
as well as the 1.90 A resolution structure of EIZS in a complex
with four Hg2þ ions in an unusual closed active site conformation
˚
and the 1.90 A resolution structure of ligand-free D99N EIZS in
an open active site conformation. Comparisons of these struc-
tures provide new insight regarding the active site closure
mechanism of bacterial terpenoid cyclases. Furthermore, muta-
genesis of aromatic residues in the EIZS active site results in the
production of new sesquiterpene product arrays because of the
remolded template for FPP cyclization as well as altered modes of
stabilization of carbocation intermediates. Accordingly, we also
˚
present the 1.64 A resolution structure of the F198A EIZS-
Mg2þ3-PPi-BTAC complex.
MATERIALS AND METHODS
Expression and Purification of Epi-Isozizaene Synthase
(EIZS). Recombinant EIZS from S. coelicolor A3(2) was
expressed at high levels in Escherichia coli BL21(DE3) and
purified as previously described (13) with minor modifications.
Briefly, E. coli BL21(DE3) carrying pET28a(þ)/SCO5222 was
inoculated into Luria-Bertani (LB) medium containing kanamy-
cin and grown overnight at 37 ꢀC. A total of 4 L of LB/
kanamycin medium was inoculated with 5 mL of the overnight
seed culture, and E. coli was grown at 37 ꢀC until the OD600
reached 0.5. The temperature was reduced to 20 ꢀC, and the cells
Crystallization and Determination of the Structure of the
EIZS-Mg2þ3-PPi-BTAC Complex. EIZS was crystal-
lized by the sitting drop vapor diffusion method. Typically, a
4 μL drop of protein solution [8-10 mg/mL EIZS, 20 mM Tris-
HCl (pH 7.5), 300 mM NaCl, 10 mM MgCl2, 10% glycerol,
2 mM TCEP, 2 mM sodium pyrophosphate, and 2 mM
benzyltriethylammonium chloride] was added to 4 μL of pre-
cipitant solution [100 mM Bis-Tris (pH 5.5), 25-28% polyethy-
lene glycol 3350, and 0.2 M (NH4)2SO4] and equilibrated against
a 1 mL well reservoir of precipitant solution. Crystals appeared
within 2-3 days and grew to maximal dimensions of 100 μm ꢀ
were induced with 0.1 mM isopropyl β-D-thiogalactopyranoside
for 18 h. Cells were harvested, resuspended in 50 mL of Talon
buffer A [50 mM sodium phosphate (pH 8.0), 300 mM NaCl,
20% glycerol, and 5 mM β-mercaptoethanol (BME)], supple-
mented with phenylmethanesulfonyl fluoride and DNase, and
sonicated for 6 min using a 40% duty cycle and 30% power
range. After three cycles of sonication, the cell lysate was clarified
by centrifugation at 16000g and 4 ꢀC for 75 min. The clarified
lysate was loaded on a Talon Co2þ metal affinity resin (5 mL),
and a step gradient from 0 to 200 mM imidazole in Talon buffer
was applied to elute the enzyme. The fractions were analyzed
using SDS-PAGE, and the most concentrated fractions were
pooled and applied to a Superdex gel filtration column equili-
brated in 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10%
glycerol, 10 mM MgCl2, and 2 mM tris(2-carboxyethyl)pho-
sphine (TCEP). The fractions were analyzed by SDS-PAGE,
and the fractions containing the enzyme were pooled and
concentrated to 8 mg/mL enzyme using a YM-10 centricon.
The resulting protein preparation was >99% pure on the basis of
SDS-PAGE.
˚
10 μm ꢀ 10 μm. Crystals diffracted X-rays to 1.60 A resolution at
the Advanced Photon Source (Argonne, IL), beamline NE-CAT
24-ID-C, and belonged to space group P21 with one monomer in
3
˚
the asymmetric unit, a Matthews coefficient (VM) of 2.1 A /Da,
corresponding to a solvent content of 43%, and the following
˚
˚
˚
unit cell parameters: a = 53.185 A, b = 47.374 A, c = 75.376 A,
and β = 95.53ꢀ.
Heavy-atom derivatives were prepared by soaking crystals in
precipitant buffer supplemented with 1 mM ethylmercury chlor-
ide for 1-2 days. Diffraction data were collected at the absorp-
˚
tion edge of mercury (1.0548 A) on beamline X29 at the National
Synchrotron Light Source (NSLS). Crystals diffracted X-rays to
˚
1.90 A and belonged to space group P21 with the following unit
˚
˚
˚
cell parameters: a = 51.693 A, b = 46.549 A, c = 75.551 A, and
β = 92.34ꢀ. Data reduction was achieved with Denzo and
Scalepack (17). Difference Patterson maps generated with anom-
alous scattering data revealed large peaks corresponding to
mercury. The initial electron density map was phased by single-
wavelength anomalous dispersion (SAD) of mercury using
autoSHARP (18). Following density modification and automatic
building, approximately 50% of the protein residues were built
Site-Directed Mutagenesis. Single site-specific mutations
were introduced into the EIZS wild-type plasmid using primers 1
and 2 for each respective mutant as follows (lowercase letters

Article
Biochemistry, Vol. 49, No. 8, 2010 1789
Table 1: Data Collection and Refinement Statistics
EIZS-Mg2þ3-PPi-BTAC
complex
EIZS-Hg2þ
complex
F198A EIZS-Mg2þ3-PPi-BTAC
D99N EIZS
(ligand-free)
4
complex
Data Collection
˚
wavelength (A)
1.0080
1.0548
1.075
0.9795
˚
resolution (A)
40-1.60
46113
50-1.90
28446
50-1.64
45831
50-1.90
28694
no. of unique reflections
completenessa (%)
redundancya
92.9 (100)
3.6 (3.6)
99.4 (98.6)
5.1 (5.1)
99.8 (100)
3.6 (3.5)
0.062 (0.238)
97.6 (95.6)
3.3 (3.0)
0.089 (0.634)
a,b
Rsym
0.058 (0.208)
0.075 (0.325)
Refinement
c
R
cryst/Rfree
0.158/0.189
0.169/0.202
0.156/0.190
0.162/0.207
˚
rmsd for bonds (A)
rmsd for angles (deg)
rmsd for dihedral angles (deg)
no. of atoms
0.012
1.4
0.005
0.8
0.015
1.6
0.009
1.1
21
15
18
16
protein
2858
431
55
2512
335
13
2858
467
31
2638
316
5
solvent
ligand
Ramachandran plot (%)
allowed
94.7
5.3
95.2
4.8
95.0
5.0
94.4
5.6
additionally allowed
P
P
b
aValues in parentheses refer to the highest-resolution shell. Rsym
=
|Ih - ÆIhæ|/ Ih, where ÆIhæ is the average intensity over symmetry equivalent
P
P
c
refections. Rcryst
=
||Fobs| - |Fcalc||/ |Fobs|, where summation is over the data used for refinement. Rfree was calculated as described for Rcryst by using 5%
of the data excluded from refinement.
into the electron density map. At this point, the molecular model
of EIZS was refined against the 1.60 A resolution data set
C213, C243, and C283. Iterative cycles of refinement and manual
model building were achieved with PHENIX and COOT,
respectively. Ions and water molecules were included in later
cycles of refinement. Individual atomic B factors were utilized,
and the mercury, chloride, and sulfur atoms were refined
anisotropically. Data reduction and refinement statistics are
listed in Table 1. A total of 323 of 381 residues are present in
the final model, as there was no interpretable density for the
A251-L267 loop as well as the N- and C-termini.
˚
collected from the native crystal instead of the mercury deriva-
tive. Iterative cycles of refinement and manual model building
using CNS (19), O (20), COOT (21), and PHENIX (22) allowed
for the assembly of the complete protein model. Individual
atomic B factors were utilized during refinement. Buffer mole-
cules, ions, water, glycerol, the benzyltriethylammonium cation
(BTAC), PPi, and a sulfate ion (hydrogen bonded to R163, H164,
R220, and R226) were included in later cycles of refinement. Data
reduction and refinement statistics are listed in Table 1. A total of
340 of 381 residues (A16-N355) are present in the final model, as
the N- and C-termini are disordered.
Determination of the Crystal Structure of Ligand-Free
D99N EIZS. The D99N EIZS mutant was crystallized by the
hanging drop vapor diffusion method. Crystals formed with a
precipitant solution at a pH slightly higher than that of the wild-
type crystals [100 mM Bis-Tris (pH 6.0), 25-28% polyethylene
glycol 3350, and 0.2 M (NH4)2SO4] and were improved by
successive rounds of microstreak seeding using D99N crystals
Determination of the Crystal Structure of the F198A
EIZS-Mg2þ3-PPi-BTAC Complex. The F198A EIZS
mutant was crystallized by the hanging drop vapor diffusion
method with the same conditions used to crystallize the wild-type
enzyme, but with successive rounds of microstreak seeding
using native crystals as the seed stock. Crystals diffracted to
˚
as the seed stock. Crystals diffracted to 1.9 A resolution at APS
beamline NE-CAT 24-ID-C and belonged to space group
˚
P212121 with the following unit cell parameters: a = 41.144 A,
˚
˚
˚
1.64 A resolution at NSLS beamline X29 and belonged to space
b = 81.952 A, and c = 106.693 A. Molecular replacement
calculations were performed with PHASER using the atomic
coordinates of native EIZS (less ligand and solvent atoms) as a
search probe. Iterative cycles of refinement and manual model
building were achieved with PHENIX and COOT, respectively.
Sulfate, glycerol, and water molecules were included in later
cycles of refinement. Individual atomic B factors were utilized.
Data reduction and refinement statistics are listed in Table 1. A
total of 316 of 381 residues (P18-E335) are present in the final
model, as the N- and C-termini are disordered.
˚
group P21 with the following unit cell parameters: a = 53.241 A,
˚
˚
b = 47.179 A, c = 75.568 A, and β = 95.57ꢀ. Molecular replace-
ment calculations were performed with PHASER (23) using the
atomic coordinates of native EIZS (less ligands and solvent
atoms) as a search probe. The electron density clearly revealed
the F198A mutation. Iterative cycles of refinement and manual
model building were achieved with PHENIX and COOT, respec-
tively. Ions, PPi, BTAC, and water molecules were included in later
cycles of refinement. Individual atomic B factors were utilized.
Determination of the Crystal Structure of the EIZS-
Hg2þ4 Complex. To investigate the mercury binding sites of the
ethylmercury chloride-derivatized crystals used for structure
Determination of Kinetic Parameters of EIZS Mutants.
EIZS mutants were assayed as previously described (13) in 50
mM piperazine-N,N0-bis(2-ethanesulfonic acid) (PIPES) (pH
6.5), 20% glycerol, 100 mM NaCl, 10 mM MgCl2, and 5 mM
BME. Each series of assays was performed two or three times
using concentrations of [1-3H]FPP (100 mCi/mmol) ranging
˚
determination, refinement of the 1.90 A resolution structure
was completed. Four mercury binding sites were identified by
strong peaks in a Bijvoet difference Fourier map adjacent to C68,

1790 Biochemistry, Vol. 49, No. 8, 2010
Aaron et al.
F
IGURE 1: (a) Ribbon plot of the EIZS-Mg2þ3-PPi-BTAC complex showing the aspartate-rich motif (red) and the NSE motif (orange)
flanking the mouth of the activesite. The Mg2þ ionsare shown as magenta spheres, and PPi and BTAC molecules are color-coded by atom (yellow
for carbon, blue for nitrogen, red for oxygen, and orange for phosphorus). Helices are labeled according to the convention first established for
FPP synthase (12). (b) The quaternary ammonium group of the benzyltriethylammonium cation (BTAC) serves as a mimic of a carbocation
intermediate in catalysis.
from 0.025 to 50 μM. The optimal enzyme concentration for each
mutant was determined, where the dependence of product
formation on enzyme concentration was linear and less than
10% of the substrate was turned over: wild type (1 nM), F96A,
F198A, and W203F (20 nM). A 1 mL reaction mixture in a test
tube was overlaid with 1 mL of hexane immediately after addition
of substrate, covered with aluminum foil, and incubated for
15 min at 30 ꢀC. The reaction was quenched by addition of 75 μL
of 500 mM EDTA (pH 8.0) and the mixture vortexed for 20 s.
The hexane extract was passed through a silica gel column
directly into a scintillation vial containing 5 mL of scintillation
fluid. The aqueous phase was extracted with an additional 2 ꢀ
1 mL of hexane and passed through the same silica gel column.
Finally, the column was washed with an additional 1 mL of
hexane. A Beckman scintillation counter was used to measure
product formation, and the substrate concentration versus rate of
product formation data were fit by nonlinear regression using
Prism to determine kcat based on the known total enzyme
concentration.
Analysis of Product Arrays Generated by EIZS Mu-
tants. Substrate farnesyl diphosphate (60 μM) was incubated
with 40 μM mutant EIZS (F96A, F198A, or W203F) in 6 mL of
buffer (50 mM PIPES, 15 mM MgCl2, 100 mM NaCl, 20%
glycerol, and 5 mM BME) and overlaid with 3 mL of HPLC-
grade n-pentane in a glass test tube at 30 ꢀC for 18 h. Reaction
products were extracted with n-pentane three times, dried with
anhydrous MgSO4, and concentrated on an ice/water mixture
under reduced pressure until the volume was reduced to 100 μL.
The products were analyzed using an Agilent 6890 GC/JEOL
JMS-600H mass spectrometer, using a 30 m ꢀ 0.25 mm HP5MS
capillary column (Department of Chemistry, Brown University)
in EI (positive) mode using a temperature program of 60-280 ꢀC,
with a gradient of 20 ꢀC/min and a solvent delay of 3.5 min.
Analysis of the organic extracts resulting from the incubation of
FPP with the mutant cyclases by gas chromatography and mass
spectrometry (GC-MS) reveals the formation of mixtures of
sesquiterpene hydrocarbons with m/z 204. Compounds were
identified by comparison of their individual mass spectra and
chromatographic retention indices with those of authentic com-
pounds in the MassFinder 3.0 Database (24).
highest-resolution structure of any terpenoid cyclase determined
to date. EIZS adopts the class I terpenoid synthase R-helical
fold and consists of a bundle of 10 R-helices, designated A-J
˚
(Figure 1a), in which the 20 A deep active site is defined mainly by
helices C, D, G, H, and J. Among the terpenoid cyclases of
known structure, EIZS is structurally most similar to pentalenene
˚
synthase (rmsd of 3.3 A for 304 CR atoms). EIZS crystallizes as a
monomer, consistent with dynamic light scattering measure-
ments indicating that the protein is a monomer in solution
(data not shown).
The electron density map clearly reveals three Mg2þ ions, PPi,
and a BTAC molecule bound in the active site (Figure 2a; for
reference, the molecular structure of BTAC is illustrated in
Figure 1b). The side chain of D99 in the aspartate-rich
D99DRHD103 motif coordinates to Mg2þ and Mg2þ with
A
C
syn,syn-bidentate geometry, while Mg2þ is chelated by the
B
NSE N240DLCSLPKE248 motif (bold indicates Mg2þ ligands).
Each Mg2þ ion is coordinated with octahedral geometry, with
nonprotein coordination sites occupied by the oxygen atoms of
PPi and by water molecules. The PPi anion also accepts hydrogen
bonds from the side chains of R194, K247, R338, and Y339
(Figure 2b). It is likely that similar metal coordination and
hydrogen bond interactions are normally formed with the dipho-
sphate group of the substrate FPP in the precatalytic Michaelis
complex. These interactions stabilize the closed active site con-
formation that sequesters FPP from bulk solvent and triggers
ionization to initiate the electrophilic cyclization cascade.
Interestingly, the detailed structural changes that occur be-
tween open and closed active site conformations differ between
fungal and plant terpenoid cyclases. For example, the rmsd
between ligand-free and trichodiene synthase from Fusarium
sporotrichioides in a complex with Mg2þ and PPi is 1.4 A (9),
˚
3
whereas that of bornyl diphosphate synthase from Salvia offici-
˚
nalis (culinary sage) is only 0.6 A for the catalytic domain (25).
Until now, the structure of the closed conformation of a bacterial
terpenoid cyclase has remained unknown because the only other
bacterial terpenoid cyclase crystal structure, that of pentalenene
synthase, had been determined only in the ligand-free state (6).
Importantly, the residues that interact with Mg2þ ions or PPi in
EIZS are conserved in pentalenene synthase. Superposition of the
closed, ligand-bound structure of EIZS with ligand-free pentale-
nene synthase reveals a very similar alignment of the metal
binding motifs, with pentalenene synthase helices D and H being
RESULTS AND DISCUSSION
Structure of the EIZS-Mg2þ3-PPi-BTAC Complex.
˚
˚
At 1.60 A resolution, the crystal structure of this complex is the
1.5 A farther apart than in EIZS. We hypothesize that upon

Article
Biochemistry, Vol. 49, No. 8, 2010 1791
F
IGURE 2: (a) Simulated annealing omit maps (black) of the PPi anion, Mg2þ ions, and BTAC, contoured at 5σ. Note the cation-π interactions
between the positively charged quaternary ammonium group of BTAC and the aromatic rings of F95, F96, and F198 (red dashed lines). (b) Metal
coordinationinteractions(black dashed lines) and hydrogenbondinteractions(red dashedlines) inthe EIZS-Mg2þ3-PPi complex. (c) Simulated
annealing omit maps (black) of the PPi anion, Mg2þ ions, and BTAC in the active site of F198A EIZS, contoured at 5σ. Note the alternative
position of BTAC resulting from the F198A mutation.
binding of Mg2þ and substrate or PPi, the active site of
D100N mutant has lost >95% of its activity compared to the
native enzyme (14), suggesting that the D100N mutation disrupts
the D100-R338-PPi hydrogen bond network. In trichodiene
synthase, the second aspartate in the aspartate-rich motif, D101,
similarly stabilizes a hydrogen bond network with R304 and
PPi (9), suggesting that the bacterial and fungal cyclases share the
same molecular strategy for linking the molecular recognition of
the substrate diphosphate group with the mechanism of active
site closure.
Similar analysis of the steady-state kinetic parameters of
pentalenene synthase mutated in the aspartate-rich motif (27)
has established that the first aspartate in the DDLFD segment
has the largest effect on catalytic activity, with the D80E
substitution resulting in an ∼3500-fold reduction in kcat/KM.
By contrast, the D81E substitution results in a more modest 400-
fold reduction in kcat/KM (27). Notably, the D84E substitution
results in an only ∼3-fold reduction in kcat/KM, consistent with
the finding that in EIZS the corresponding third aspartate in the
DDxxD motif points away from the active site and is not involved
3
pentalenene synthase undergoes a change to a closed conforma-
tion comparable to that observed for the EIZS-Mg2þ
PPi-BTAC complex.
-
3
Although the first and third aspartate residues in the aspartate-
rich metal binding motif coordinate to Mg2þA and Mg2þC in the
plant terpenoid cyclases containing a complete trinuclear metal
cluster such as (þ)-bornyl diphosphate synthase (25), limonene
synthase (26), and (þ)-δ-cadinene synthase (11), only the first
aspartate residue in this motif coordinates to Mg2þA and Mg2þ
C
in complexes of the fungal cyclases trichodiene synthase (9) and
aristolochene synthase (10) with Mg2þ3 and PPi. EIZS is similar
to the fungal cyclases in that only D99 coordinates to Mg2þA and
Mg2þC. A critical role for D99 in metal complexation is reflected
in the dramatic losses of catalytic activity measured for the D99N
and D99E mutants (14). Although D100 of EIZS does not
directly interact with the Mg2þ ions or PPi, it does accept a
hydrogen bond from R338, which also donates a hydrogen bond
to PPi (Figure 2b). Site-directed mutagenesis reveals that the

1792 Biochemistry, Vol. 49, No. 8, 2010
Aaron et al.
F
IGURE 3: Mg2þ3-PPi binding motif that is conserved among the bacterial and fungal sesquiterpene cyclases otherwise related by <20% amino
acid sequence identity: (a) EIZS from S. coelicolor A3(2) (PDB entry 3KB9), (b) aristolochene synthase from Aspergillus terreus (PDB entry
2OAB, monomer D), and (c) trichodiene synthase from F. sporotrichioides (PDB entry 1JFG, monomer B).
in substrate binding. Thus, the structure of the EIZS-Mg2þ
-
3
in all other ligand-free terpenoid cyclase structures. Instead, helix
G, which forms one side of the active site (and contains R194, a
residue that donates a hydrogen bond to PPi in the ligand-bound
structure), bends by ∼110ꢀ to occupy the location formerly
occupied by the Mg2þ3-PPi binding motif (Figure 4b). Addi-
PPi-BTAC complex, as well as mutagenesis results with both
EIZS (14) and pentalenene synthase (27), suggests that the third
aspartate in the DDxxD motif of bacterial terpenoid cyclases
is not involved in metal binding or essential for catalysis.
Remarkably, the molecular recognition of Mg2þ3-PPi is con-
served among fungal cyclases such as aristolochene synthase and
trichodiene synthase, and bacterial cyclases, such as EIZS, that
otherwise bear no significant overall amino acid sequence
relationship (Figure 3).
˚
˚
tionally, helix D moves ∼1.5 A outward, helix J moves ∼4 A
outward, while helix H, which was bent in the ligand-bound
structure to enable Mg2þB chelation by the NSE motif, becomes
straight and also moves outward (Figure 4a). The positions and
conformations of helices C, E, and F remain essentially un-
changed, and there is no interpretable electron density for helix I
or the loop (A251-L267) connecting it to helices H and J. Salt
bridges between helix G and helices D and F stabilize the closed
ligand-free structure: R194 makes a salt bridge with E175
(helix F), and R195 makes a salt bridge with D103 (helix D)
(Figure 4b). Although these residues are found in pentalenene
synthase as R173, F174, D84, and E152, a comparable closed
ligand-free conformation was not observed by Lesburg and
colleagues (6). The role of the Hg2þ ions in dislodging the
Mg2þ3-PPi-BTAC binding motif to result in the closed ligand-
free conformation of EIZS is unclear; inspection of the mercury
binding sites does not reveal any particular structural changes that
would trigger the observed change in the conformation of helix G.
To further explore structural changes resulting from the
dissociation of the Mg2þ3-PPi-BTAC binding motif from the
active site of EIZS, we determined the structure of D99N EIZS at
Crystals of EIZS form upon addition of BTAC (Figure 1b),
commonly used in synthetic organic chemistry as a phase transfer
catalyst. As a hydrophobic cation, BTAC mimics the bisabolyl
carbocation intermediate proposed for the EIZS-catalyzed cycli-
zation mechanism (13). The positively charged quaternary am-
monium group of BTAC appears to be stabilized by long-range
electrostatic interactions with the PPi anion (the shortest N-O
˚
separation is 4.1 A), as well as cation-π interactions with the
aromatic side chains of F95, F96, and F198 (N-ring centroid
˚
separations range from 4.7 to 5.4 A) (Figure 2a). Such cation-π
interactions are proposed to play a critical role in stabilizing the
highly reactive carbocation intermediates found in all enzyme-
catalyzed terpenoid cyclizations (6, 28, 29). The binding of BTAC
in the active site of EIZS demonstrates that the bacterial
terpenoid cylase active site can accommodate and stabilize a
positively charged ligand resembling the positively charged
carbocation intermediates of the normal cyclization cascade.
˚
1.9 A resolution. This amino acid substitution compromises the
Although determined at a much lower resolution of 2.85 A, the
syn,syn-bidentate coordination of Mg2þA and Mg2þC (Figure 2b)
and consequently diminishes catalytic efficiency (14). The crystal
structure reveals the complete absence of the Mg2þ3-PPi-
BTAC binding motif in the active site. Moreover, the active site
of D99N EIZS adopts an open conformation (Figure 4c) without
any conformational changes in helix G. Therefore, we conclude
that the conformational change of helix G shown in panels a and
b of Figure 4 is somehow caused by Hg2þ binding. Comparison
of the structures of ligand-free D99N EIZS and the
EIZS-Mg2þ3-PPi-BTAC complex reveals ligand-induced con-
formational changes in helix H and loop H-R-1, as well as the
J-K loop (which is completely disordered in D99N EIZS)
(Figure 4c). The overall rmsd between wild-type and D99N EIZS
˚
recently reported structure of the trichodiene syntha-
se-Mg2þ3-PPi-BTAC complex provides an example of the
stabilization of positively charged ligands in the active site of a
fungal terpenoid cyclase (30).
Structures of the EIZS-Hg2þ Complex and D99N
4
EIZS. To date, we have been unable to prepare crystals of
wild-type EIZS in a completely ligand-free state, since additives
BTAC and PPi required for crystallization are not readily
dialyzed out of the crystals. However, soaking crystals of the
EIZS-Mg2þ3-PPi-BTAC complex with ethylmercury chloride
displaces three Mg2þ ions, PPi, and BTAC to yield the 1.90 A
˚
resolution structure of the EIZS-Hg2þ complex in which all
4
Hg2þ ions bind remotely from the active site (Figure 4a). In this
complex, Hg2þA is coordinated by C68, Hg2þB is coordinated by
C283 (the electron density is best interpreted as two alternate
positions), Hg2þ is coordinated by C243, and Hg2þ is co-
structures is 1.6 A for 318 CR atoms.
˚
Structure of the F198A EIZS-Mg2þ3-PPi-BTAC
Complex. To investigate structural changes in the active site
resulting from mutagenesis of aromatic residues, we determined
C
D
˚
ordinated by C213 (two alternate positions). Surprisingly, how-
ever, the now ligand-free active site is not open and empty, as it is
the X-ray crystal structure of F198A EIZS at 1.64 A resolution.
This mutant was selected for X-ray crystallographic study

Article
Biochemistry, Vol. 49, No. 8, 2010 1793
F
IGURE 4: (a) Superposition of ribbon plots of the EIZS-Mg2þ3-PPi-BTAC (green) and EIZS-Hg2þ4 (cyan; Hg2þ ions appear as red spheres)
complexes. Helices D, G, H, and J, which undergo the largest changes, are labeled. (b) Salt bridges (black dashes) between helix G and helices
D and F stabilize the closed ligand-free structure. (c) Superposition of the EIZS-Mg2þ3-PPi-BTAC complex (green) and D99N EIZS (purple),
illustrating the structural changes in helix H and the H-R-1 and J-K loops that accompany active site closure.
because it retains some catalytic activity (Table 2) and exhibits a
Table 2: Steady-State Kinetic Parameters for Wild-Type EIZS and Site-
remarkably altered product array (Table 3) (vide infra). Overall,
Specific Mutants
the F198A mutation results in minimal structural perturbations,
protein
k
cat (s-1
)
KM (nM)
kcat/KM (M-1 s-1
)
and the rmsd between the structure of wild-type EIZS and F198A
˚
WT
0.045 ( 0.003
710 ( 100
770 ( 130
1200 ( 200
1450 ( 240
(6.3 ( 0.1) ꢀ 104
310 ( 60
250 ( 45
EIZS is 0.10 A for 340 CR atoms. In the active site, the largest
structural changes resulting from the F198A substitution are
an ∼30ꢀ rotation of the side chain of F95 and an alterna-
tive rotamer of M73. The binding mode of the Mg2þ3-PPi
binding motif is identical to that observed in the wild-type
enzyme; however, the BTAC molecule occupies an alternative
position such that its benzyl ring makes quadrupole-
quadrupole interactions with the aromatic rings of F95,
F96, W203, and W325 (Figure 2c). Surprisingly, four solvent
molecules are observed in the active site, forming a hydrogen
bonding network with N233 and the backbone carbonyl
of A236.
F96A
F198A
W203F
0.00024 ( 0.00002
0.00030 ( 0.00002
0.00034 ( 0.00003
230 ( 40
Activity of Aromatic Mutants and Altered Product
Arrays. Mutagenesis of residues that coordinate to metal ions
or donate hydrogen bonds to the substrate diphosphate group
can significantly compromise catalysis by a terpenoid cyclase.
For example, mutation of EIZS metal-binding residues can result
in a >95% decrease in cyclase activity, as well as altered ratios of
aberrant cyclization products (14). Amino acid substitutions that

1794 Biochemistry, Vol. 49, No. 8, 2010
Aaron et al.
Table 3: Distribution of Sesquiterpene Products for Wild-Type EIZS and Site-Specific Mutants
relative product percentage (%)
protein unknown sesquisabine β-farnesene
epi-
zizaene
β-
Z-R-
sesqui-
Z-γ-
unknown unknown unknown unknown
R-
A
isozizaene
acoradiene bisabolene phellandrene bisabolene
B
C
D
E
cedrene
WT
-
7
2
9
5
70
5
79
8
9
-
-
7
-
-
6
1
-
-
7
-
-
-
6
-
-
7
-
-
7
-
-
-
3
2
-
-
-
F96A
F198A
W203F
-
12
-
6
20
7
-
14
13
24
47
4
6
-
-
-
8
F
IGURE 5: (a) Stereoview of the active site surface contour encapsulated by the closed conformation of EIZS shown as a magenta mesh. The
aspartate-rich motif (red) and the NSE motif (orange) are oriented as in Figure 1. (b) Cyclization product, epi-isozizaene, modeled into the
enclosed active site contour of EIZS (magenta mesh), and the location of the Mg2þ3-PPi cluster is shown as a visual reference. (c) Enclosed active
site contour of F198A EIZS (light brown meshwork) into which the new cyclization product β-acoradiene is modeled. The remolded active site
contour in this mutant prevents epi-isozizaene formation but permits the formation of new or alternative sesquiterpene products predominantly
derived from the bisabolyl carbocation intermediate.
change the contour and electronic environment of the active site
can alter the product profile of a terpenoid cyclase even more
dramatically. The crystal structure of EIZS reveals that the active
site contour is defined by hydrophobic and aromatic residues
that, when locked in the closed conformation by three Mg2þ ions
and the substrate diphosphate group, create a unique template
that preferentially chaperones the cyclization of FPP to form epi-
isozizaene (Figure 5). The active site contour appears to be
product-like in shape, although it does not appear to be as
complementary a fit as, for example, that between the closed
active site contour of the A. terreus aristolochene synthase and its
sesquiterpene product (10). This is a possible signal of catalytic
promiscuity, in that wild-type EIZS generates 80% epi-isozizaene
and 20% of a mixture of alternative sesquiterpene products (14).
While a less-perfect template can result in the formation of
multiple products, the active site contour of a terpenoid cyclase
can be further remolded by structure-based site-directed muta-
genesis to permit greater product diversity.
have prepared the F96A, F198A, and W203F mutants. These
residues were selected for mutation on the basis of their proximity
to the BTAC cation (Figure 2). Steady-state kinetic parameters
measured for these mutants (Table 2) indicate that the mutations
only modestly affect KM but more significantly affect kcat
.
Accordingly, overall catalytic efficiencies (kcat/KM) are decreased
205-275-fold. Notably, none of the three active site aromatic
mutants generate epi-isozizaene as a major product; indeed, no
epi-isozizaene whatsoever is generated by the F198A mutant, and
the active site contour of this variant as observed in the crystal
structure of the F198A EIZS-Mg2þ3-PPi-BTAC complex is
more complementary in shape to bisabolene-derived cyclization
products such as β-acoradiene, as illustrated in Figure 5. Nota-
bly, β-acoradiene is not generated by wild-type EIZS, so the
appearance of this spiroterpenoid represents a new catalytic
activity introduced by a single amino acid substitution. Altering
the active site contour of EIZS alters the conformations and
cyclization trajectories of FPP and reactive carbocation inter-
mediates, and the formation of alternative products appears to
depend on how well a particular alternative product fits the
As the first step in exploring the importance of active site
aromatic residues for catalysis and product diversity in EIZS, we

Article
Biochemistry, Vol. 49, No. 8, 2010 1795
F
IGURE 6: Biosynthetic versatilityofEIZScan bemanipulated bysite-directed mutagenesis, asillustrated for sesquiterpene productsidentified for
wild-type (WT) and mutant cyclases. In general, more diverse sesquiterpene product arrays result from the substitution of aromatic residues
definingthe activesitecontour(red labels) thanfromsubstitutionofresiduesthat coordinate the Mg2þ ionsrequiredfor catalysis [bluelabels(14)].
For example, F198A EIZS does not generate epi-isozizaene at all but instead generates a mixture of sesquisabinene A, Z-R- and Z-γ-bisabolenes,
sesquiphellandrene, and β-acoradiene as its major cyclization products. Remolding the active site contour permits the generation of alternative
products as long as they can be accommodated within the remolded template, as illustrated for β-acoradiene in Figure 5. It is interesting to note
that many of these reactions have been examined in theoretical and computational studies (33).
remolded active site contour in a mutant cyclase. Additionally,
the F96A and F198A substitutions could compromise the
potential stabilization of carbocation intermediates by cation-π
interactions. Some of the sesquiterpenes generated by these
aromatic mutants have previously been observed as side products
generated by both wild-type and mutant EIZS enzymes (14),
while three sesquiterpene products have not previously been
observed with this cyclase (Figure 6 and Table 3).
(E)-β-Farnesene, the major product (70%) formed by F96A
EIZS, results from deprotonation at C-31 of the allylic cation that
results from the initial ionization of FPP; other reaction products
include sesquisabinene A, Z-γ-bisabolene, and an unidentified
hydrocarbon presumed to be a sesquiterpene based on mass
spectrometry, with m/z 204. F198A EIZS generates sesquisabi-
nene A, (E)-β-farnesene, zizaene, β-acoradiene, Z-R-bisabolene,
sesquiphellandrene, Z-γ-bisabolene, and three unidentified ses-
quiterpenes. Interestingly, F198A EIZS generates no epi-isozi-
zaene. The predominant product of W203F EIZS is Z-γ-
bisabolene, generated by the abstraction of a proton from the
intermediate bisabolyl cation (Figure 6). Epi-isozizaene accounts
for 14% of the products of the W203F mutant; the remaining
products include sesquisabinene, (E)-β-farnesene, zizaene, and
four unidentified sesquiterpenes. It is notable that epi-isozizaene
biosynthetic activity is preserved, if only partially so, in the EIZS
mutant with the most conservative aromatic-aromatic substitu-
tion, which presumably preserves more of the general contour
and electrostatic profile of the active site.
conformational changes that occur upon ligand binding in
bacterial sesquiterpene cyclases. In the closed, ligand-bound
conformation, only the first aspartate of the D99DRHD motif
coordinates to Mg2þ and Mg2þC; substitution of a neutral
A
asparagine residue for the negatively charged aspartate residue at
position 99 is sufficient to disrupt the assembly and stability of the
trinuclear metal cluster required for substrate recognition and
activation. Conformational changes that accompany active site
closure in the bacterial cyclase involve helix H and the H-R-1
loop, and the J-K loop at the C-terminus (Figure 4c). Corre-
sponding structural changes generally accompany active site
closure in fungal (31) and plant (25) terpenoid cyclases. The
template for FPP cyclization is fully formed in the closed active
site conformation of EIZS as well as all other class I terpenoid
cyclases.
In general, the permissiveness and promiscuity of terpenoid
cyclases vary, both in terms of the substrates they accept and the
product(s) they generate. These properties are dictated by the
three-dimensional contour of the fully formed template in the
closed active site conformation. Many terpenoid cyclases, such as
A. terreus aristolochene synthase (32), are high-fidelity cyclases
that generate one product exclusively. However, other cyclases
such as EIZS generate one major product and (usually) minor
quantities of one or more alternative products (14). The
structural basis for such mechanistic promiscuity is presum-
ably rooted in how well the active site contour enforces the
correct regiochemistry and stereochemistry for cyclization
and eventual quenching of the carbocation intermediates by
chaperoning the conformations of reactive intermediates.
Intriguingly, the conformations and orientations of such
intermediates may not reflect the original conformation and
orientation of the substrate if the template is somewhat
permissive (33). A more permissive template allows alterna-
tive premature quenching of on-pathway intermediates or
CONCLUDING REMARKS
The crystal structures of the EIZS-Mg2þ3-PPi-BTAC com-
plex and D99N EIZS are particularly informative in that they
reveal two active site conformations: a closed, ligand-bound
conformation and an open, ligand-free conformation, respec-
tively. Comparison of these structures reveals, for the first time,

1796 Biochemistry, Vol. 49, No. 8, 2010
Aaron et al.
off-pathway conformations that lead to the formation of
aberrant products.
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from the active site (35). With EIZS, two different strategies have
been employed to manipulate the cyclization template. Mutagen-
esis of metal-binding residues appears to have an only modest
effect on the cyclization template such that the fidelity of epi-
isozizaene biosynthesis is not significantly compromised. Indeed,
certain amino acid substitutions involving the Mg2þ-binding
residues, such as D100N, N240D, S244A, and E248D, actually
lead to increased proportions of epi-isozizaene and lower levels of
the alternative sesquiterpene products, although with signifi-
cantly decreased overall catalytic efficiency (Figure 6) (14).
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or the derived cations (and which therefore contribute directly to
the active site contour) significantly compromises the fidelity of
epi-isozizaene biosynthesis (Figure 6), with the F198A substitu-
tion completely suppressing epi-isozizaene formation and redir-
ecting the cyclization cascade toward the generation of alter-
native acyclic, monocyclic, and bicyclic sesquiterpenes. Thus,
remolding the active site contour by mutagenesis opens up new
cyclization trajectories while closing off old ones.
The appearance of low levels of new or alternative cyclization
products resulting from mutagenesis of the active site contour in a
terpenoid cyclase may reflect past or future evolutionary poten-
tial; i.e., catalytic promiscuity in enzyme function may provide a
“toehold of evolution” (36). The evolution of biosynthetic
diversity in this family of enzymes is achieved by simply remold-
ing the active site contour to promote one cyclization pathway
while suppressing hundreds of others, and it is notable that this is
readily achieved by only a handful of amino acid substitutions.
This work represents the first step in deciphering the relationship
between the structure of the EIZS active site and its biosynthetic
specificity as a product-like template for terpenoid cyclization
reactions: the three-dimensional contour of the active site can be
remolded to better fit another product and disfavor others, even
to the point of excluding epi-isozizaene formation. That the
biosynthetic specificity of a terpenoid cyclase is so sensitive to and
so readily manipulated by minimal mutagenesis in nature or in
the laboratory will likely contribute to the growing structural and
stereochemical diversity of the terpenome.
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We thank the National Synchrotron Light Source at Brookhaven
National Laboratory (beamline X29A) and the Advanced Photon
Source at Argonne National Laboratory (NE-CAT beamline
24-ID-C) for access to X-ray crystallographic data collection
facilities. We thank Dr. Wayne Chou for providing FPP and
assistance with GC-MS experiments and Cristina Virgilio for
assistance with protein preparation.
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