Substrate specificities of E- and Z-farnesyl diphosphate synthases with substrate analogs

Article abstract of DOI:10.1016/j.molcatb.2012.04.006

Prenyltransferases catalyzes the basic isoprenoid chain elongation to produce prenyl diphosphates, which led to upward of 30,000 diverse isoprenoids as steroids, carotenoids, natural rubbers, and prenyl proteins. Here, we determined the reactivities of E- and Z-farnesyl diphosphate synthases (E- and Z-FPP synthases) isolated from Bacillus stearothermophilus and Thermobifida fusca, respectively. For this purpose we use the synthetic substrate analogs, 8-tetrahydropyran-2-yloxy-, 8-hydroxy- and 8-acetoxygeranyl diphosphates. Z-FPP synthase catalyzed the reaction between 8-hydroxygeranyl diphosphate (HOGPP) and isopentenyl diphosphate (IPP), which produced (2Z)-12-hydroxyfarnesyl diphosphate (yield: 16.7%) and (2Z, 6Z)-16-hydroxygeranylgeranyl diphosphate (yield: 6.6%). Neither E- nor Z-farnesyl diphosphate synthases detectably catalyzed reactions between 8-tetrahydropyran-2-yloxygeranyl diphosphate (8-THPOGPP) and IPP. However, a mutated E-FPP synthase (Y81S), did catalyze this reaction, producing 12-tetrahydropyran-2-yloxyfarnesyl diphosphate (12-THPOFPP) with a yield of 12.3%. Wild-type E-FPP synthase catalyzed the reaction of 8-acetoxygeranyl diphosphate (8-AcOGPP) with IPP, which produced 12-acetoxyfarnesyl diphosphate (12-AcOFPP) (yield, 21.8%). Mutant E-FPP synthase catalyzed the reaction between 8-AcOGPP with IPP, producing 12-AcOFPP and 16-acetoxygeranylgeranyl diphosphate (16-AcOGGPP) with respective yields of 55.3% and 1.7%. We believe our results contribute to a better understanding of the catalytic properties of these key enzymes and illustrate their use in the stereo-specific syntheses of compounds that may have significant biotechnological and medical applications.

Full text of DOI:10.1016/j.molcatb.2012.04.006

Journal of Molecular Catalysis B: Enzymatic 80 (2012) 1–6
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Substrate specificities of E- and Z-farnesyl diphosphate synthases with substrate
analogs
a,∗
a
a
a
a
Masahiko Nagaki , Takumi Ichijo , Rikiya Kobashi , Yusuke Yagihashi , Tohru Musashi ,
a
b
c
d
Jun Kawakami , Norimasa Ohya , Takeshi Gotoh , Hiroshi Sagami
Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan
Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Kojirakawa-machi, Yamagata 990-8560, Japan
Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata Gakuen-cho, Akita 010-8502, Japan
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, Japan
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Prenyltransferases catalyzes the basic isoprenoid chain elongation to produce prenyl diphosphates, which
led to upward of 30,000 diverse isoprenoids as steroids, carotenoids, natural rubbers, and prenyl pro-
teins. Here, we determined the reactivities of E- and Z-farnesyl diphosphate synthases (E- and Z-FPP
synthases) isolated from Bacillus stearothermophilus and Thermobifida fusca, respectively. For this purpose
we use the synthetic substrate analogs, 8-tetrahydropyran-2-yloxy-, 8-hydroxy- and 8-acetoxygeranyl
diphosphates. Z-FPP synthase catalyzed the reaction between 8-hydroxygeranyl diphosphate (HOGPP)
and isopentenyl diphosphate (IPP), which produced (2Z)-12-hydroxyfarnesyl diphosphate (yield: 16.7%)
and (2Z, 6Z)-16-hydroxygeranylgeranyl diphosphate (yield: 6.6%). Neither E- nor Z-farnesyl diphosphate
synthases detectably catalyzed reactions between 8-tetrahydropyran-2-yloxygeranyl diphosphate (8-
THPOGPP) and IPP. However, a mutated E-FPP synthase (Y81S), did catalyze this reaction, producing
12-tetrahydropyran-2-yloxyfarnesyl diphosphate (12-THPOFPP) with a yield of 12.3%. Wild-type E-FPP
synthase catalyzed the reaction of 8-acetoxygeranyl diphosphate (8-AcOGPP) with IPP, which produced
Received 14 November 2011
Received in revised form 5 April 2012
Accepted 5 April 2012
Available online 15 April 2012
Keywords:
E-farnesyl diphosphate synthase
Z-farnesyl diphosphate synthase
Substrate specificity
Hydroxygeranyl diphosphate
Tetrahydropyran-2-yloxygeranyl
diphosphate
Acetoxygeranyl diphosphate
1
2-acetoxyfarnesyl diphosphate (12-AcOFPP) (yield, 21.8%). Mutant E-FPP synthase catalyzed the reac-
tion between 8-AcOGPP with IPP, producing 12-AcOFPP and 16-acetoxygeranylgeranyl diphosphate
16-AcOGGPP) with respective yields of 55.3% and 1.7%. We believe our results contribute to a better
(
understanding of the catalytic properties of these key enzymes and illustrate their use in the stereo-
specific syntheses of compounds that may have significant biotechnological and medical applications.
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
give E- or Z-FPP as the ultimate products as shown in Fig. 2. Our
present research was undertaken to define in more detail the cat-
alytic properties of these metabolically key enzymes.
Prenyltransferases catalyze the stereospecific condensation
between isopentenyl diphosphate (IPP) and an allylic diphosphate,
thereby generating over 30,000 diverse isoprenoids. These com-
pounds serve as precursors in the biosynthesis of steroids and
carotenoids, and serve to modify quinones and proteins as shown
in Fig. 1 [1–5]. The prenyltransferases are divided into two main
classes, E- and Z-condensing, and can be subdivided further into
three groups based on whether they catalyze the production of
short-, medium-, or long-chain products as shown in Table 1 [6–8].
E- and Z-farnesyl diphosphate synthases (FPP synthases), respec-
tively catalyze the E- and Z-condensation of IPP with dimethylallyl
diphosphate (DMAPP) and/or with geranyl diphosphate (GPP) to
E-FPP synthase is widely distributed and has been purified
from a number of organisms, and the gene encoding Z-FPP syn-
thase from Mycobacterium tuberculosis was cloned and expressed
by Crick and co-workers [9]. These findings have stimulated
numerous studies focusing on Z-prenyltransferases [9–12].
We recently described the reactivities of several allylic sub-
strate analogs, such as 8-hydroxygeranyl-, 8-methoxygeranyl-,
4-methoxymethoxydimethylallyl-, and 4-vinyldimethylallyl-
diphosphates in reactions catalyzed by wild-type or mutant
Bacillus stearothermophilus E-FPP synthase [13,14]. We also deter-
mined the substrate specificities of E- and Z-FPP synthases from B.
stearothermophilus and from Thermobifida fusca, respectively [15].
Here, we extended our investigations into the substrate speci-
ficities of E-FPP synthase and Z-FPP synthase by determining their
catalytic capabilites for two kinds of allylic substrate analogs with
∗ Corresponding author. Tel.: +81 172 39 3947; fax: +81 172 39 3947.
E-mail address: nagaki@cc.hirosaki-u.ac.jp (M. Nagaki).
1
381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.006

2
M. Nagaki et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 1–6
Fig. 1. Isoprenoid biosynthesis.
Table 1
Classification of E- and Z-prenyl chain elongating enzymes.
Type
Group
Prenyl transferase
Example
E-
PE-I
PE-II
PE-III
Short-chain prenyl elongating enzyme
Medium-chain prenyl elongating enzyme
Long-chain prenyl elongating enzyme
E-FPP synthase (C15), E-GGPP synthase (C20)
HexPP synthase (C30), HepPP synthase (C35)
OctPP synthase (C40), SPP synthase (C45)
Z-
PZ-I
PZ-II
PZ-III
Short-chain prenyl elongating enzyme
Medium-chain prenyl elongating enzyme
Long-chain prenyl elongating enzyme
Z-FPP synthase (C15)
DeDolPP synthase (C80), UPP synthase (C55)
Rubber synthase (C>5000)
FPP synthase, farnesyl diphosphate synthase; GGPP synthase, geranylgeranyl diphosphate synthase; HexPP synthase, hexaprenyl diphosphate synthase; HepPP synthase,
heptaprenyl diphosphate synthase; OctPP synthase, octaprenyl diphosphate synthase; SPP synthase, solanesyl diphosphate synthase; DeDolPP synthase, dehydrodolichyl
diphosphate synthase; UPP synthase, undecaprenyl diphosphate synthase.
a hydrophilic group (hydroxyl, tetrahydropyranyloxy, or acetoxy)
at the ␻-position.
in other experiments [13–16]. Identification of the prenyl alco-
hols derived from alkaline phosphatase treatment of the reaction
products was carried out using GC–MS analysis conducted on a
JEOL Q1000GC-Mk II Mass Spectrometer (Japan Electron Optics
Laboratory Co. Ltd., Tokyo, Japan) that included an HP-5 column
2
. Materials and methods
Ø
2.1. Analysis
(30 m × 0.32 mm) using a 1 ml/min helium carrier flow rate. The
◦
temperature was programmed to deliver a 15 C/min gradient that
◦
The prenyl alcohols produced by alkaline phosphatase treat-
began with a 4.7-min hold at 50 C and ended with a 2-min hold at
◦
ment of the products generated by the enzymatic reactions were
measured using an HPLC system consisting of the following com-
ponents: Hitachi type L-6000 pump, Hitachi L-7420 (LaChrom)
UV–VIS detector, Chromato-DAQ II A/D converter (Ulvac Inc., Chi-
280 C. Injector and GC-transfer line temperatures were both set to
◦
200 C [16].
The yields of the FPP synthase reactions were determined rel-
ative to those of FPP derived from reactions using IPP and GPP as
substrates. The IR spectra were acquired using a Hitachi 260-10 and
a BIO-RAD FTS-30. The nuclear magnetic resonance (NMR) spectra
were acquired using JEOL JNMGX 270 FT and JEOL JNM-ECA 500 FT
®
gasaki, Japan) and a LiChroCART (Merck) column (Merck KGaA,
Darmstadt, Germany). Compounds were eluted with hexane:2-
propanol (40:1) mixtures, which are similar to those we used

M. Nagaki et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 1–6
3
Fig. 2. Z (or E)-farnesyl diphosphate synthase-catalyzed reactions between allylic substrate analogs and IPP.
NMR spectrometers using trimethylsilane in CDCl3 as an internal
standard.
tetrabutylammonium fluoride (TBAF)/acetic anhydride in tetrahy-
drofuran yielding 8-acetoxygeraniol (8-AcOGOH, yield; 0.79 g,
9
8.7%). 1H NMR (500 MHz, CDCl , TMS): ı 1.65 (3H, s), 1.66 (3H,
3
2
.2. Synthesis of substrate analogs
s), 2.07 (3H, s), 2.18 (2H, q, J = 7.3 Hz), 4.13 (2H, d, J = 6.8 Hz),
.45 (2H, s), 5.39 (1H, t, J = 6.3 Hz) and 5.44 (1H, t, J = 6.6 Hz). 13
NMR (125 MHz, CDCl3, MS): ı 13.9, 16.2, 21.0, 25.9, 38.9, 59.1,
4
C
2
.2.1. Synthesis of 8-hydroxygeranyl diphosphate
−
1
8
-Hydroxygeranyl diphosphate (HOGPP) was synthesized
70.3, 124.1, 130.2, 138.3, and 171.2. IR (KBr, cm ): 3300, 2860,
according to our published method [13]. Briefly, 8-hydroxygeranyl
acetate which was obtained by acetylation of geraniol, was oxidized
with titanium dioxide in tertiary butylhydroperoxide (t-BuOOH)
and 8-hydroxygeranyl chloride which was obtained by the chlori-
nation of the acetate, was phosphorylated by Davisson’s method
1700, and 1250. GC retention time was 13.54 min. GC–MS: m/z
212 [M]+ (rel. int. 0.6%), 194 [M−18] (34.2%), 152 [M−60] (1.4),
+
+
+
+
134 [M−60−18] (34.7), 84 (base peak), and 67 [M−60−18−67]
(21.4). Acetoxygeranyl diphosphate (AcOGPP) was prepared from
AcOGOH using similar methods [13].
[
17].
2.3. Purification of E- and Z-FPP synthases
2
.2.2. Synthesis of 8-tetrahydropyran-2-yloxygeranyl
diphosphate
Hydroxygeranyl acetate (HOGOAc, yield: 2.34 g, 3.4%) was pre-
2.3.1. Purification of wild type and mutated B.
stearothermophilus E-FPP synthases
pared by oxidation of geranyl acetate (4.06 g) with selenium dioxide
in the presence of t-BuOOH. Geranyl acetate was synthesized
by acetylation of geraniol (5.01 g). Subsequently, the hydroxyl
group of HOGOAc (1.0 equiv.) was treated with dihydropyrane
in the presence of 0.1 equiv. of pyridinium-p-toluene sulfonate.
The reaction yielded 8-tetrahydropyran-2-yloxygeranyl acetate (8-
THPOGOAc). THPO-geraniol (yield: 2.71 g) was then obtained by
hydrolysis of THPO-geranyl acetate (3.40 g) with potassium car-
The purification of wild-type E-FPP synthase from B. stearother-
mophilus ATCC 10149 was carried out according to our published
methods [8,13–15,18–21]. We purified the mutated E-FPP synthase
(Y81S) from Escherichia coli DH5␣ cells harboring the plasmids
encoding the corresponding mutated FPP synthases [19]. The cells
were harvested, suspended in 50 mM Tris–HCl (pH 7.0), 10 mM 2-
mercaptoethanol, 1 mM EDTA, and disrupted by sonication with
◦
two pulses at 40% power at 0 C using a model 250 Advanced Bran-
bonate in methanol [13]. 1H NMR (CDCl , TMS): ı 1.66 (3H, s),
son sonifier (Branson Ultrasonics Corporation, Kanagawa, Japan).
3
◦
1
.67 (3H, s), 1.53–1.86 (6H, m), 2.08 (2H, t, J = 7.2 Hz), 2.17 (2H,
q, J = 6.7 Hz), 3.85–4.09 (2H, d, J = 11.5 Hz), 4.13 (2H, d, J = 6.8 Hz),
.61 (1H, s), and 5.40 (2H, t, J = 6.3 Hz). 13C NMR (70 MHz, CDCl3,
TMS): ı 13.9, 16.0, 19.2, 25.3, 25.7, 30.4, 38.9, 59.1, 61.8, 72.2, 96.8,
The homogenate was heated at 55 C for 60 min and centrifuged at
100,000 × g for 10 min. Further purification of the mutated enzyme
4
was carried out according to our published method [13].
−1
1
1
24.8, 128.0, 132.0, 139.0. IR (KBr, cm ): 3400, 2850, 1700, and
100. The GC retention time was 12.47 min. GC–MS of the alcohol:
2.3.2. Purification of Z-FPP synthase of T. fusca
The expression plasmid pMluUEX [7,23,24], kindly provided
by Prof. Tanetoshi Koyama of Tohoku University, was used to
transform E. coli BL21(DE3) (TaKaRa Bio Inc., Shiga, Japan). Transfor-
+
+
m/z 254 [M] (obscure), 170 [M−84] (relative intensity [rel. int.]
+
+
8
.7%), 152 [M−84−18] (3.1), 134 [M−84−18−18] (20.0), 54 (base
peak). Chlorination of 8-THPO-geraniol was carried out to produce
-THPO-geranyl chloride, and the chloride was converted to the
corresponding diphosphate by Davisson’s method [17].
◦
mants were grown at 37 C as streak cultures and then transferred
8
to 20 ml of LB broth containing 100 ␮g/ml of ampicillin and cul-
◦
tured overnight at 37 C. The cultures were next transferred to 2 l
◦
of the same (fresh) medium and further incubated at 37 C until
2
.2.3. Synthesis of 8-acetoxygeranyl diphosphate
Hydroxygeranylsilyl (HOGTBDPS) ether (yield; 19.04 g, 16.4%)
reaching an absorbance of 0.6 at 600 nm (mid-log phase). Isopropyl
␤-d-thiogalactopyranoside was then added to 0.1 mM, to induce
protein expression, the cells were harvested after incubation for an
additional 12 h at 20 C and then were disrupted by sonication. The
insoluble fraction was removed by centrifugation at 6000 × g for
was prepared by oxidizing geranyl t-butyl diphenylsilyl chlo-
ride with selenium dioxide in the presence of t-BuOOH.
The 8-hydroxygeranylsilyl ether was deprotected with
◦

4
M. Nagaki et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 1–6
3
0 min. After applying the supernatant to a His TrapTM FF Column
B. stearothermophilusFPP synthase
(
GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA), the elu-
ate was precipitated with demineralized ammonium sulfate, and
the resulting protein solution (approximately 200 ␮g/ml) was used
for the enzymatic reactions. Protein concentrations were estimated
using the Bradford method with BSA as a standard [15].
N
C
8
1
86
FPPsynthase (wild type) 76 -EMIHT
Y
S
SLIH DD LPSM D -92
SLIH DD LPSM D -92
mutantFPP synthase Y81S
76 -EMIHT
2.4. Enzyme reaction conditions
Fig. 3. Sequence differences between wild-type and mutant farnesyl diphosphate
synthases.
2.4.1. E-FPP synthase reaction
The reaction mixture for B. stearothermophilus E-FPP synthase
reaction contained, in a total volume of 1 ml, consisted of 200 ␮mol
Tris–HCl (pH 8.5), 10 ␮mol MgCl , 50 ␮M ␤-mercaptoethanol
2
determined by 2D-Noesy NMR spectra, are obtained by the reaction
between GPP and 1, respectively, using E- and Z-FPP synthases [22].
We were therefore able to reasonably conclude that the product
was 2Z-12-hydroxyfarnesol (2Z-HOFOH, 3a-OH).
(
BME), 5 ␮mol KCl, 0.5 ␮mol of an allylic substrate (HOGPP or
THPOGPP), 0.5 ␮mol IPP, and wild-type or mutant E-FPP synthases
◦
(
approximately 25 ␮g), and incubated at 55 C for 3 h. The reaction
mixture was next treated with alkaline phosphatase (Roche Diag-
In contrast, the spectrum of the latter alcohol (68.3 min
on HPLC) showed an ambiguous molecular ion, however, and
◦
nostics GmbH, Mannheim, Germany) at 37 C for 5 h, extracted with
pentane, and analyzed by HPLC and GC–MS.
+
+
the dehydration ions [M−18] and [M−18−18] were observed
distinctly at m/z 288 (rel. int. 0.04%) and 270 (6.0). These val-
ues corresponded to C20H32O and C20H30, respectively, together
2.4.2. Z-FPP synthase reaction
The incubation mixture for T. fusca Z-FPP synthase reaction
contained 100 ␮mol Tris–HCl (pH 7.5), 5 ␮mol BME, 10 ␮mol
+
with additional fragment ions at m/z 203 [M−18−18−67]
+
(
2.6), 135 [M−18−18−67−68] (13.6), and 93 (base peak),
MgCl , 1.0 ␮mol of an allylic substrate (HOGPP, THPOGPP, or
2
indicating that the product possessed a structure consis-
AcOGPP), 1.0 ␮mol of IPP, and Z-FPP synthase (approximately
tent with 16-hydroxy-3,7,11,15-tetramethylhexadeca-2,6,10,14-
tetraen-1-ol (16-HOGGOH). We were therefore able to reasonably
conclude that the product was 2Z, 6Z-16-HOGGOH (4a-OH), pro-
vided that an enzymatic reaction similar to that of Z-FPP synthase
occurred [25,26].
◦
1
00 ␮g) in a total volume of 1 ml. After incubation at 37 C for
6
h, the reaction mixture was processed similarly as mentioned
above.
2
.5. Estimation of the optimized structures
The reaction between HOGPP and IPP that is catalyzed by E-
FPP synthase only produces E-HOFOH [14]. Further, using the
Y81S mutant E-FPP synthase (Fig. 3), which functions as a GGPP
synthase, the reaction with these same substrates is known
to produce minor amounts of E-hydroxygeranylgeraniol and E-
hydroxygeranylfarnesol [14]. This result therefore shows that the
substrate specificity of Z-FPP synthase with respect to HOGPP was
less restricted than that of E-FPP synthase.
The estimation of optimized structures was performed by theo-
retical calculations using the Macintosh OS version of the Chem3D
Pro 5.0 energy minimization program (Perkin Elmer, Tokyo, Japan).
This program visualizes molecules and uses the molecular mechan-
ics method (MM2) to calculate molecular energies associated with
potential conformations of model compounds such as 8-THPOFOH
and geranylgeraniol (GGOH).
3
. Results and discussion
3.2. Reaction of THOGPP (2b) with IPP (1) catalyzed by wild-type
and mutant FPP synthases recombinant enzymes
Here, to compare the substrate specificities of E- and Z-FPP syn-
thases, we examined the reaction between HOGPP and IPP that
was catalyzed by Z-FPP synthase, as we had already previously
characterized the reaction between HOGPP and IPP catalyzed by E-
FPP synthase [13]. We also examined the reactivities of THPOGPP
and AcOGPP, which possess, respectively, a THPO or AcO group
at the ␻-position. It is important to note that the yields of the
FPP synthase reactions were determined relative to those of FPP
derived from reactions using IPP and GPP as substrates, as shown
in Table 2.
No products were detected in the reaction between (2b)
and (1) with E- or Z-FPP synthases. In contrast, Y81S FPP syn-
thase generated a product from these same to substrates that
was hydrolyzed to an alcohol by alkaline phosphatase. The alco-
hol’s retention time on HPLC was 31.3 min (relative yield, 12.3%).
After preparative HPLC, the product was analyzed by GC–MS. MS
showed an ion [M]+ at m/z 322 (rel. int. 0.004%), corresponding
+
to C20H34O , together with fragment ions at m/z 304 [M−18]
3
+
+
(0.002), 220 [M−18−84] (1.8), 202 [M−18−102 (pyranosyloxy)]
+
(
24.0), 134 [M−18−102−68] (8.7%), and 55 (base peak). These
values indicated that the product possessed a 12-tetrahydropyran-
-yloxyfarnesol (THPOFOH) structure. We therefore decided to
3.1. Reaction between HOGPP (2a) and IPP (1) catalyzed by
2
Z-FPP synthase
designate the product as E-THPOFOH, 5b-OH, taking into account
the mechanism of the FPP synthase reaction [25,26].
The hydrolysates derived from the product of the Z-FPP syn-
thase reaction between 2a and 1 eluted as two peaks on HPLC at
However, when the optimized the structures of THPOFOH, 5b-
OH, and GGOH were calculated via the MM2 method, the length
8
2.2 min and 68.3 min, which were subjected to GC–MS. The mass
of a THPO group (3.61 A˚ ) was almost equivalent to that of one
spectrum of the former (82.2 min on HPLC) showed a molecular
isoprene unit (3.91 A˚ ). We therefore concluded that 5b-OH is a
ion at m/z 238 (rel. int. 0.02%), corresponding to C15H26O , and
2
+
GGOH-type product, based on its length. Further, since the mutant
E-FPP synthase functions as a GGPP synthase, the synthase reaction
proceeded as expected. The substition of Y81 by a less bulky seryl
residue relieves the steric hindrance due to the Y81 benzyl group,
thereby allowing a GGPP-type product to be synthesized [13].
the main ion fragments were observed at m/z 220 [M−18] (1.6),
+
+
2
02 [M−18−18] (15.8), 135 [M−18−18−67] (7.9), and 93 (base
peak), indicating that the product possessed a 12-hydroxy-3,7,11-
trimethyldodeca-2,6,10-trien-1-ol (12-hydroxyfarnesol) structure
as shown in Fig. 2. We have already shown that E- and Z-farnesol, as

M. Nagaki et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 1–6
5
Table 2
Relative yields of the reaction products.
Allylic substrate
Product (%)a
HOFOH
HOGGOH
THPOFOH
n.d.
AcOFOH
AcOGGOH
Z-FPP synthase reaction + IPP
HOGPP
THPOGPP
16.7 (3a-OH)
6.6 (4a-OH)
E-FPP synthase reaction + IPP
HOGPP
11.2b (5a-OH)c
THPOGPP
AcOGPP
n.d.
21.8 (5c-OH)
55.3b (5c-OH)
Mutated E-FPP synthase (Y81S) + IPP
HOGPP
THPOGPP
AcOGPP
12.9b (6a-OH)c
12.3 (5b-OH)
1.7b (6c-OH)
n.d., not detected.
The relative yields of product alcohols derived from reactions catalyzed by FPP synthase were relative to the yields of FOH derived from the corresponding enzymatic
reaction of GPP with IPP. Abbreviations: HOFOH, hydroxyfarnesol; HOGGOH, hydroxygeranylgeraniol; THPOFOH, tetrahydropyran-2-yloxyfarnesol; AcOFOH, acetoxyfarnesol;
AcOGGOH, acetoxygeranylgeraniol.
a
As described in Section 2, reaction products were converted to alcohols for analysis.
Values calculated from GC data.
The values indicated the data which we reported by Ref. [14].
b
c
3
.3. Reactions of acetoxy-GPP (2c) with IPP (1) catalyzed by
6Z)-16-hydroxygeranylgeranyl diphosphate. This finding provides
new insight into the catalytic potential of Z-FPP synthase.
Neither E- nor Z-FPP synthase catalyzed the reaction between
8-THPOGPP and IPP. However, a mutant E-FPP synthase (Y81S)
produced 12-THPOFPP from the substrates 8-THPOGPP and IPP,
and wild-type E-FPP synthase catalyzed the reaction betweeen
8-AcOGPP and IPP producing 12-AcOFPP. Mutant E-FPP synthase
catalyzed the production of 12-AcOFPP and 16-AcOGGPP from the
reactants 8-AcOGPP and IPP. Whether or not Z-FPP synthase can
catalyze the reaction between AcOGPP and IPP will be the subject
of future research.
Since their initial isolation over a decade ago [12], Z-farnesyl
diphosphate synthases have received surprisingly little attention
despite their central role of in metabolism. Our present findings
regarding the unique substrate specificity of one representative
bacterial enzyme, particularly when compared with that of a bacte-
rial E-FPP synthase, clearly warrant additional enzymatic studies as
well as comparisons with enzymes isolated from a variety of other
organisms.
wild-type and mutant E-FPP synthases
3
.3.1. Wild-type E-FPP synthase
The alcohols derived from alkaline phosphatase treatment of
the product of the reaction of acetoxy-GPP (2c) with 1 yielded
a peak with an HPLC retention time of 30.8 min. The MS spec-
trum of the product showed an ion at m/z 280 (rel. int. 0.1%),
corresponding to C17H28O , with main fragment ions at m/z 262
3
+
+
+
[
M−18] (0.7), 202[M−18−60] (0.4), 135[M−18−60−67] (7.8),
and 93 (base peak). These data indicated that the product possesses
a 12-acetoxy-3,7,11-trimethyldodeca-2,6,10-trien-1-ol (acetoxy-
farnesol, AcOFOH) structure. The data strongly indicated that the
prenylalcohol was (all-E)-acetoxy-FOH, 5c-OH. The relative yield
was 21.8% based on the product derived from the reaction between
the natural substrates, GPP and IPP.
3
.3.2. Mutant E-FPP synthase
Mutant E-FPP synthase catalyzed the reaction of 2c with 1 yield-
ing two products, which were then hydrolyzed with phosphatase
to the corresponding alcohols for analysis. The GC retention times
of the two product alcohols were 17.8 min (HPLC, 30.8 min; rela-
tive yield: 55.3%) and 19.2 min (HPLC, ambiguous; relative yield:
Acknowledgement
This work was supported in part by a grant from the Cosmetol-
ogy Research Foundation (to M. N.).
1
.7%). The product with the 17.8 min retention time was identified
as 5c-OH.
The mass spectrum of the second species (19.2 min) showed
References
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3
+
+
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