Sesquiterpene Synthases from Coprinus cinereus
340 nm. Microplate assays were carried out with pyrophosphate re-
agent (50 mL), assay buffer (90 mL), and varying concentrations of
different substrates (10 mL). Blank reactions without substrate were
run in parallel. Assay mixtures were allowed to equilibrate for
final oven temperature of 2008C. Mass spectra were scanned in
the range of 5–300 atomic mass units at 1 s intervals.
For chiral GC–MS analysis of b-bisabolene, racemic b-bisabolene
was kindly provided by Prof. Degenhardt and used as an authentic
standard for comparison with Cop4 and Cop6 reaction products
(Figure 1). Enantiomers were assigned by comparison with the b-
bisabolene present in Bergamot essential oil, which contains only
(6S)-b-bisabolene (16s), as described by Kçllner et al.[38] The abso-
lute configuration of germacrene D (7s) synthesized by Cop4 was
determined by comparison with germacrene D enantiomers from
the essential oil of Solidago canadensis. In this essential oil, the (+)
enantiomer is more abundant (Figure S4).[72] Amyris balsamifera es-
sential oil contains only one enantiomer of cadina-4,11-diene (14s)
with known absolute configuration.[47] Cadina-4,11-diene (14s) in
this essential oil was therefore used as reference compound to de-
termine the absolute configuration of the cadina-4,11-diene (14s)
that was synthesized by Cop4 with (Z,E)-FPP as the substrate (Fig-
ure S6). Finally, the absolute configuration of the Cop6 reaction
product a-cuprenene (3s) was indirectly determined by compari-
son of its oxidation product, a-cuparene with a synthetic standard
compound containing 98% (+)-a-cuparene and 2% (À)-a-cupar-
ene. Dauben and Oberhꢃnsli[73] reported the isolation and synthesis
of cuprenenes that under retention of absolute configuration
slowly convert into the corresponding, aromatic cuparenes after
prolonged air exposure. In vitro reactions of Cop6 with (E,E)-FPP as
a substrate were therefore left standing for up to 30 d at 308C
with periodic analysis of products formed. Over time, the ring-oxi-
dized a-cuparene accumulated (Figure S3).
5 min at 308C prior to the addition of enzyme (5 mL; 0.2 mgmLÀ1
)
to start the reaction. The activity was determined as the difference
between the decrease of absorbance per minute of the sample
and of the blank. By using an extinction coefficient for NADH of
e340 nm =6.22ꢁ103 mÀ1 mLÀ1, one unit of activity was defined as the
amount of enzyme needed to release 1 mmol of PPi, inducing the
consumption of 2 mmol of NADPH. The Km and Vmax values were de-
termined by using a nonlinear fit of V versus [S] plot. The analysis
was carried out by running a macro in Xcel 2007.
In vitro analysis of sesquiterpene product profiles: Sesquiter-
pene product profiles of Cop4, Cop6 and NS1 were analyzed by in-
cubating purified enzyme (20 mL; 0.1 mgmLÀ1 in the case of Cop6
and 0.2 mgmLÀ1 in the case of Cop4 and NS1) in terpene synthase
buffer (180 mL) containing one of the four prenyl diphosphate sub-
strates investigated ((E,E)-FPP, (Z,E)-FPP, (Æ)NPP, and (E)-GPP) to
yield a final assay concentration of 100 mm. Reactions were carried
out in a glass vial for 18 h at 258C before the headspace of the
glass vial was sampled for 10 min by solid-phase microextraction
(SPME) by using a 100 mm polydimethlysiloxane fiber from (Su-
pelco/Sigma–Aldrich). After 10 min absorption, the fiber was insert-
ed into the injection port of a GC–MS for thermal desorption.
To measure the influence of reaction conditions on the product
profiles of Cop4 and Cop6 with 100 mm (E,E)-FPP as the substrate,
the terpene synthase buffer was modified by the addition of NaCl
or KCl (final assay concentration: 1m) or substitution of 10 mm
MgCl2 with 10 mm MnSO4. The pH of the reactions was changed
by substituting 10 mm Tris–HCl in the terpene synthase buffer with
10 mm of sodium carbonate (pH 10.0) or 10 mm of sodium acetate
(pH 5.0) buffer. Reactions were carried out for 18 h at 25, 4, and
378C prior to the analysis of sesquiterpene hydrocarbon products
as described above.
Structural modeling of Cop4 and Cop6 sesquiterpenes synthas-
es: Structural models in the open, unligated conformation were
built by using the structure of trichodiene synthase from F. sporotri-
choides[52] (PDB ID: 1JFA, chain A) for Cop6 (44% amino acid se-
quence similarity) and of aristolochene synthase from A. terreus
(PDB ID: 2E40, chain D)[20] for Cop4 (39% amino acid sequence sim-
ilarity). Crystal structures of trichodiene (PDB ID: 2Q9z, chain B)[48]
and of aristolochene synthase (PDB ID: 2A6, chain D)[20] in the
closed formation, ligated with Mg2+ and pyrophosphate (PPi), were
used to build the corresponding models for Cop6 and Cop4.
Models were built by using the alignment mode of the Swiss
Model homology-modeling server .[74] This method assesses protein
structures by using 3D profiles. Structures are validated by compar-
ison of an atomic model with its amino acid sequence and assign-
ment of positive (good compatibility) or negative scores for each
amino acid position. Models generated in this study have very
good compatibility scores. Protein models were visualized and
aligned with their template structure by using PyMol 0.99 devel-
oped by DeLano Scientific LLC (San Francisco, CA). Active site vol-
umes were calculated with CASTp[75] (by using the CASTpyMol ver-
sion 2.0).
Gas chromatography–mass spectrometry (GC–MS) analysis: GC–
MS analysis was carried out on a HP GC 7890 A coupled to anion-
trap mass spectrometer HP MSD triple axis detector (Agilent Tech-
nologies). Separation was carried out by using a HP-1MS capillary
column (30 mꢁ0.25 mm, i.d.: 1.0 mm) with an injection port tem-
perature of 2508C and helium as a carrier gas. Mass spectra were
recorded in electron-impact ionization mode. Volatile compounds
adsorbed on a fiber from the enzyme reaction headspace were
desorbed for 10 min in the injection port. The temperature pro-
gram started at 608C and ramped up 88CminÀ1 to a final oven
temperature of 2508C. Mass spectra were scanned in the range of
5–300 atomic mass units at 1 s intervals.
For product identification, the retention index (RI) of each com-
pound peak was determined by calibrating the GC–MS first with a
C8–C40 alkane mix. Retention indices and mass spectra of com-
pound peaks were compared to reference data in MassFinder’s
(software version 3) terpene library.[47] In addition, essential oils
with known terpene compositions were used as authentic stand-
ards as described in Table S1.
Abbreviations: FPP, farnesyl diphosphate (also-pyrophosphate);
NPP, nerolidyl diphosphate; GPP, geranyl diphosphate; PPi, pyro-
phosphate.
Absolute configuration determination: To determine the absolute
configuration of several sesquiterpenes described in this study, we
used chiral GC–MS analysis for comparison of retention times with
reference compounds. Sesquiterpenes were separated on a Quiral
b-cyclodextrin column (25 mꢁ0.25 mmꢁ0.125 mm; Chirasil-Dex,
Varian Inc.) by using a temperature program that started at 408C
for 2 min followed by ramping the temperature at 38CminÀ1 to a
Acknowledgements
This research was supported by the National Institute of Health
Grant GM080299 (to C.S.D.).We thank Prof. Jçrg Degenhardt
from the Max Planck Institute for Chemical Ecology, Jena, Germa-
ny, for his gift of the b-bisabolene standard.
ChemBioChem 2010, 11, 1093 – 1106
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1105