Subunit composition of hinokiresinol synthase controls enantiomeric selectivity in hinokiresinol formation

Article abstract of DOI:10.1039/b918656e

Asparagus officinalis hinokiresinol synthase (HRS) is composed of two subunits, HRSα and HRSβ. Individually, each subunit forms (E)-hinokiresinol (EHR) from 4-coumaryl 4-coumarate, whereas a mixture of both subunits forms (Z)-hinokiresinol (ZHR) from the same substrate. In this study, we analyzed the enantiomeric compositions of ZHR and EHR formed after incubation of 4-coumaryl 4-coumarate with recombinant subunit proteins, recHRSα and/or recHRSβ, and with naturally occurring A. officinalis ZHR. The enantiomeric composition of ZHR formed by the mixture of recHRSα and recHRSβ was (+)-100% enantiomer excess (e.e.), identical to that of A. officinalis ZHR. In contrast, the enantiomeric compositions of EHR formed by recHRSα and recHRSβ, individually, were (-)-20.6 and (-)-9.0% e.e., respectively. These results clearly demonstrate that the subunit composition of A. officinalis HRS controls not only cis/trans isomerism but also enantioselectivity in hinokiresinol formation. The Royal Society of Chemistry.

Full text of DOI:10.1039/b918656e

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Subunit composition of hinokiresinol synthase controls enantiomeric
selectivity in hinokiresinol formation
Masaomi Yamamura,a Shiro Suzuki,b Takefumi Hattoria and Toshiaki Umezawa*a,b
Received 9th September 2009, Accepted 24th November 2009
First published as an Advance Article on the web 6th January 2010
DOI: 10.1039/b918656e
Asparagus officinalis hinokiresinol synthase (HRS) is composed of two subunits, HRSa and HRSb.
Individually, each subunit forms (E)-hinokiresinol (EHR) from 4-coumaryl 4-coumarate, whereas a
mixture of both subunits forms (Z)-hinokiresinol (ZHR) from the same substrate. In this study, we
analyzed the enantiomeric compositions of ZHR and EHR formed after incubation of 4-coumaryl
4-coumarate with recombinant subunit proteins, recHRSa and/or recHRSb, and with naturally
occurring A. officinalis ZHR. The enantiomeric composition of ZHR formed by the mixture of
recHRSa and recHRSb was (+)-100% enantiomer excess (e.e.), identical to that of A. officinalis ZHR.
In contrast, the enantiomeric compositions of EHR formed by recHRSa and recHRSb, individually,
were (-)-20.6 and (-)-9.0% e.e., respectively. These results clearly demonstrate that the subunit
composition of A. officinalis HRS controls not only cis/trans isomerism but also enantioselectivity in
hinokiresinol formation.
Introduction
Norlignans are found in many coniferous trees (especially in
heartwood) and some monocotyledonous plants.1,2 Norlignans
have a diphenylpentane carbon structure and have various bio-
logical activities. For example, both (Z)- and (E)-hinokiresinols,
ZHR and EHR, which are phytoalexins of Asparagus officinalis
and Cryptomeria japonica, respectively, have various additional
bioactivities; ZHR has angiogenic inhibitor activity,3 estrogen-
like activity,4 antioxidant activity and antiatherogenic activity,5
whereas EHR has antifungal activity6 and estrogen-like activity,4
and inhibits cyclic AMP phosphodiesterase.7
Fig. 2 Biosynthetic pathway of hinokiresinols.
and HRSb. The mixture of both recombinant subunit proteins
(recHRSa and recHRSb) catalyzed the formation of ZHR from 4-
coumaryl 4-coumarate, whereas incubation of the same substrate
with only one type of subunit (recHRSa or recHRSb) gave rise to
EHR (Fig. 3). This result indicates that the subunit composition of
A. officinalis HRS can control cis/trans isomerism in hinokiresinol
formation. In addition to cis/trans isomerism, hinokiresinols
Fig. 1 The structure of hinokiresinols.
exhibit enantioisomerism due to the C7 asymmetric carbon
Hinokiresinol has the simplest norlignan structure (Fig. 1),
suggesting that this is the first compound in the norlignan
biosynthetic pathway. Enzymatic conversion of 4-coumaryl 4-
atom. ZHR from the rhizome of Anemarrhena asphodeloides
showed (-)-54% enantiomer excess (e.e.),4 while EHR from the
heartwood of Chamaecyparis obtusa was suggested to be an
coumarate into ZHR and EHR was detected using A. officinalis
optically pure (-)-isomer by calculating the amplitude of the cir-
cular dichroism (CD) spectrum of tetrahydrohinokiresinol (THR)
derived from C. obtusa EHR.4 These results strongly suggest
that hinokiresinol-producing plants have distinct mechanisms for
controlling enantiomeric selectivity in hinokiresinol formation, as
well as controlling cis/trans isomerism.
In this study, we show that the subunit composition of A.
officinalis HRS controls enantioselectivity and cis/trans isomerism
in hinokiresinol formation.
and C. japonica cell-suspension cultures, respectively8,9 (Fig. 2).
Recently, Suzuki et al. isolated cDNAs encoding hinokiresinol
synthase (HRS) from A. officinalis cell-suspension cultures.10
The A. officinalis HRS was composed of two subunits, HRSa
aResearch Institute for Sustainable Humanosphere, Kyoto University, Uji,
Kyoto 611-0011, Japan
bInstitute of Sustainability Science, Kyoto University, Uji, Kyoto, 611-0011,
Japan
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Fig. 3 HRS subunit compositions control cis/trans selectivity in hi-
nokiresinol formation.
Results
Enantiomeric relationships between (+)- and (-)-hinokiresinols and
(+)- and (-)-tetrahydrohinokiresinols
Initially, we synthesized racemic ( )-THRs by catalytic hydro-
genation of racemic ( )-EHRs, which were prepared as described
by Lassen et al.11 The obtained enantiomers of ( )-THRs were
separated on a chiral column. The first peak on the chiral high-
performance liquid chromatography (HPLC) chromatogram was
the (-)-enantiomer {retention time (tR) = 10.8 min} and the second
peak (tR = 11.2 min) was the (+)-enantiomer (Fig. 4A).
Fig. 4 The chiral HPLC chromatograms of tetrahydrohinokiresinols
(THRs). A, ( )-THRs derived from ( )-EHRs. B, THR derived from
ZHR, which was isolated from A. officinalis cells. C, THR derived from
ZHR, which was formed by the enzymatic reaction using A. officinalis
enzyme preparation. D, THR derived from ZHR, which was formed by
recHRSa+recHRSb. E, THR derived from EHR, which was formed by
recHRSa. F, THR derived from EHR, which was formed by recHRSb.
G, THR derived from EHR, which was isolated from C. japonica cells. H,
THR derived from EHR, which was isolated from the heartwood of C.
obtusa.
The absolute configuration at C7 in (-)-EHR isolated from
heartwood of C. obtusa has been assigned as S based on its optical
rotatory dispersion12 and the enantioselective synthesis of its di-
O-methylether.13 In addition, C7 in (+)-ZHR has an S config-
uration, as determined by CD spectroscopy4 and vibration CD
spectroscopy.11 On the basis of this information on hinokiresinol
enantiomers, we determined the enantiomeric composition of
THR derived from C. obtusa EHR by chiral HPLC with a
UV detector. By hydrogenation, (-)-(7S)-EHR isolated from C.
obtusa heartwood gave optically pure (+)-(7S)-THR (Fig. 4H and
Table 1G). In addition, it was reported that (-)-(7R)-ZHR gave (-)-
(7R)-THR.4 Together, the stereochemical correlation between (E)-
and (Z)-hinokiresinols and their hydrogenation product, THR,
were established as shown in Fig. 5.
isolated from A. officinalis and ZHR formed by the mixture of
recombinant subunit proteins, recHRSa+recHRSb. THR derived
from ZHR, which was isolated from A. officinalis, was an optically
pure (+)-isomer (Fig. 4B and Table 1A), indicating that A.
officinalis produces enantiomerically pure (7S)-ZHR. Similarly,
THR derived from ZHR, which was formed by the A. officinalis
enzyme preparation, was (+)-97.2% e.e. (Fig. 4C and Table 1B).
THR derived from ZHR, which was formed by the mixture of
recHRSa and recHRSb, was also an optically pure (+)-isomer
(Fig. 4D and Table 1C). In contrast, the enantiomeric composition
of THR derived from EHR, which was formed by the action of
Enantiomeric compositions of enzymatically formed hinokiresinols
and those isolated from plants
Having established the stereochemical correlation between the
hinokiresinols, EHR and ZHR, and their tetrahydro derivative,
THR, we next determined enantiomeric compositions of ZHR
Table 1 Enantiomeric composition of hinokiresinols
Hinokiresinol
THR
Entry
Origin
Z/E
Predominant enantiomer
Enantiomeric composition (% e.e.)
A
B
C
D
E
F
G
A. officinalis cells (isolated)
A. officinalis cells (enzyme)
recHRSa + recHRSb
recHRSa
Z
Z
Z
E
E
E
E
(+)-S
(+)-S
(+)-S
(+)-S
(+)-S
(+)-S
(+)-S
100
97.2
100
20.6
9.0
83.3
100
recHRSb
C. japonica cells (isolated)
C. obtusa heartwood (isolated)
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product.10 Here, we show that the subunit composition determines
the enantiomeric composition of hinokiresinols. Incubation of
4-coumaryl 4-coumarate with the mixture of equal amounts of
recHRSa and recHRSb resulted in an optically pure (7S)-isomer
of ZHR (Fig. 4D, Fig. 6 and Table 1C). This isomer is identical
to that isolated from A. officinalis cells (Fig. 4B and Table 1A).
A similar result was obtained with the plant protein from A.
officinalis; ZHR formed from the same substrate with the plant
protein was 97.2% e.e., that is, almost optically pure (Fig. 4C and
Table 1B). Conversely, when each subunit protein, recHRSa or
recHRSb, was individually incubated with the same substrate,
EHR was the sole product.10 Interestingly, the enantiomeric
compositions of EHR thus formed were 20.6 and 9.0% e.e. in
favor of the (7S)-enantiomer (Fig. 4E, F, Fig. 6 and Table 1D,
E). Taken together, these results clearly indicate that the subunit
composition of HRS controls not only cis/trans selectivity but
also enantioselectivity in hinokiresinol formation (Fig. 4 and
Fig. 6).
Fig. 5 An enantiomeric correlation between (E)- and (Z)-hinokiresinols
and tetrahydrohinokiresinol.
recHRSa, was (+)-20.6% e.e. (Fig. 4E and Table 1D). Similarly,
that of THR derived from EHR, which was formed by the action
of recHRSb, was (+)-9.0% e.e. (Fig. 4F and Table 1E). The
enantiomeric composition of THR derived from C. japonica EHR
was (+)-83.3% e.e. (Fig. 4G and Table 1F).
We further confirmed the enantiomeric compositions of THRs
(Fig. 4C, E, F, and G) except for optically pure THRs (Fig. 4B,
D, and H). This was performed by collecting approximately
half of each peak followed by quantification using GC-MS, so
that the contamination by the antipodes was eliminated. Based
on the quantification, enantiomeric compositions of THRs were
estimated as follows: THR derived from ZHR formed by A.
officinalis enzyme preparation, (+)-95% e.e.; THR derived from
EHR formed by recHRSa, (+)-26% e.e.; THR derived from
EHR formed by recHRSb, (+)-13% e.e.; THR derived from EHR
isolated from C. japonica cultured cells, (+)-80% e.e. Although
these enantiomeric compositions are based on the quantification
of approximate half of each peak, the % e.e. values are in
accordance with those determined by chiral HPLC with the
UV detector (Table 1). This confirms that the minor peaks of
(-)-enantiomer of THR shown in Fig. 4C and G are not due
to impurities. Thus, each HRS subunit forms both (7S)- and
(7R)-EHRs with the former as a predominant enantiomer, which
contrast sharply with the production of optically pure (7S)-ZHR
with the mixture of recHRSa and recHRSb.
Fig. 6 HRS subunit compositions control enantioselectivity in hino-
kiresinol formation.
The optically pure ZHR was isolated from A. officinalis cultured
cells. However, the enantiomeric composition of ZHR isolated
from the rhizome of A. asphodeloides was not optically pure;
(7R)-54% e.e. Similarly, EHR isolated from C. obtusa heartwood
was the optically pure (7S)-isomer, whereas EHR obtained from
C. japonica cultured cells was not optically pure; (7S)-83.3%
e.e. (Fig. 4G and Table 1F). These results indicate that the
enantiomeric compositions of hinokiresinols vary among plant
species. This may result from the different subunit compositions
of HRS of each plant species.
Together, our results show a novel example of enantiomeric
control in the biosynthesis of natural products. This dif-
fers from the enantioselectivity in the biosynthesis of lignans,
which represent another class of phenylpropanoid compounds
closely related to norlignan in terms of structure and bio-
synthesis.
Discussion
We have previously demonstrated that A. officinalis HRS is
composed of two subunits (HRSa and HRSb) and that the
subunit composition can control the cis/trans isomerism of the
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Lignans with 9(9¢)-oxygen also show a diversity of enantiomeric
compositions.2,14,15 First, dibenzylbutyrolactone lignans are opti-
cally pure, while furofuran and furan lignans are mixtures of both
enantiomers and exhibit various enantiomeric compositions. Sec-
ond, dibenzylbutyrolactone lignans are levorotatory. Third, pre-
dominant enantiomers of furofuran, furan, and dibenzylbutane
lignans vary among plant species. Fourth, the absolute configura-
tions of the predominant enantiomers of various lignans isolated
from a single plant species sometimes differ.14,15 The biosynthesis of
lignans with 9(9¢)-oxygen is initiated by the enantioselective dimer-
ization of two coniferyl alcohol units with the aid of a unique and
effective asymmetric inducer, dirigent protein (DP), to yield opti-
cally active pinoresinol (furofuran).16–21 However, the enantiomeric
composition of pinoresinol varies widely among plant species,2,14,15
suggesting that enantiomeric control by DP is not strong enough
to yield optically pure pinoresinol in planta. Additionally, the
enantiomeric compositions of lignans are determined by the first
step mediated by DP, and also subsequent metabolic steps medi-
ated by pinoresinol/lariciresinol reductase and secoisolariciresinol
dehydrogenase.14 Recently, Nakatsubo et al. demonstrated that
pinoresinol reductases (PrR, a variation of PLR in Arabidopsis
thaliana) together with a dirigent protein(s) are involved in
enantiomeric control in lignan biosynthesis in A. thaliana.22
This study demonstrated conclusively that differential expression
of PrR isoforms with distinct selectivities for substrate enan-
tiomers can determine enantiomeric composition of the product,
lariciresinol.22
The basic carbon skeleton of hinokiresinols consists of phenyl-
propane (C6C3) and phenylethane (C6C2) units that are linked via
a C7–C8¢ bond. This bond can not be formed via the coupling of
two p-coumaryl-alcohol-derived phenoxy radicals as in the case
of enantioselective lignan formation, which inevitably leads to
C8–C8¢ bonds. Hence, an enantioselective mechanism, which is
totally different from that in lignan biosynthesis, was expected
for norlignan biosynthesis. In this study, we show that this
mechanism relies on appropriate subunit composition of HRS.
That is, combining the subunits of HRS in an appropriate manner
can form ZHR with an identical enantiomeric composition
to that of the ZHR from A. officinalis (Fig. 4, Fig. 6 and
Table 1).
blotted with paper towel. The cells were weighed and stored in
liquid nitrogen until use. A cell-suspension culture of C. japonica
cv. Kumotooshi was initiated and maintained as described by
Suzuki et al.9 The cells were collected and stored in liquid nitrogen
until use.
Instrumentation
Nuclear magnetic resonance (NMR) spectra were obtained with a
JNM-LA400MK FT-NMR system (JEOL Co., Ltd.) Chemical
shifts and coupling constants (J) are expressed in d and Hz,
respectively.
GC-MS was carried out with a Shimadzu QP-5050A GC-MS
system (Shimadzu Co., Ltd.) [electron impact mode (70 eV);
column, Shimadzu Hicap CBP10-M25-025 column (Shimadzu
Co., Ltd.; 20 m ¥ 0.22 mm); carrier gas, helium; injection
temperature, 240 ◦C; column temperature (40 ◦C at t = 0 to
2 min, then to 230 ◦C at 25 ◦C min-1)]. The samples for GC-MS
were dissolved in N,O-bis(trimethylsilyl)acetamide (BSA) and left
standing at 60 ◦C for 45 min; then an aliquot of the BSA solution
was subjected to GC-MS analysis.
Reversed-phase HPLC was performed with a Shimadzu LC-6A
liquid chromatograph (Shimadzu Co., Ltd.), detection being at l =
280 nm. The reversed-phase columns used were a Waters Novapak
C18 (Waters Co., Ltd.; 3.9 mm ¥ 150 mm, 4 mm) and a YMC-Pack
Pro C18 RS column (YMC Co., Ltd.; 4.6 cm ¥ 150 mm, 3 mm).
The mobile phase used for both columns was with CH3CN–H2O
(40 : 60) at a flow-rate of 1 ml min-1.
Chiral HPLC was performed with an HPLC system comprising
a Waters 600E system controller (Waters Co., Ltd.), a Waters
60F fluid pump (Waters Co., Ltd.), and a Waters 2487 dual l
absorbance detector (Waters Co., Ltd.), detection being at l =
280 nm. The chiral column used was a Chiralpak AD column
(Daicel Chemical Co., Ltd.; 4.6 mm ¥ 150 mm, 3 mm) and the
mobile phase was EtOH at a flow-rate of 0.5 ml min-1. The sign
for optical rotation of THR enantiomers was determined using
a JASCO OR-990 chiral detector (JASCO Co., Ltd.). Silica gel
column chromatography and silica gel thin-layer chromatography
(TLC) employed Kieselgel 60 (Merck Co., Ltd.; 70–230 mesh) and
Kieselgel 60 F254 (Merck Co., Ltd.; 20 ¥ 20 cm, 0.5 or 0.25 mm),
respectively.
The control of enantioselectivity is very important from a
pharmaceutical standpoint. It is well known that each enantiomer
has different biochemical properties. Thus, the selective synthesis
of only one enantiomer is a challenging subject in the production
of chiral pharmaceuticals. In this context, the results presented
herein could provide a new approach in enantioselective organic
synthesis. Further research should be carried out to elucidate the
mechanisms that control cis/trans and enantiomeric selectivities
by HRSs. X-ray crystallographic analysis of A. officinalis HRS
may play a critical role in such research.
Chemicals
4-Coumaryl 4-coumarate was synthesized as described
previously.8 ( )-THRs were prepared as follows. ( )-EHRs,
which were synthesized exactly by the method of Lassen et al.,11
were dissolved in methanol and hydrogenated under hydrogen
with 5% palladium on carbon as a catalyst for 1.5 h at room
temperature. The mixture was filtered and concentrated to dryness
under reduced pressure. ( )-THRs: m/z [EI, THR TMS ether]
400 (M+, 19%), 385 (2), 207 (63), 179 (100); 1H-NMR (400 MHz;
CDCl3) d: 0.73 (3H, t, J = 7.3), 1.49 (1H, m), 1.64 (1H, m), 1.78
(1H, m), 1.89 (1H, m), 2.36 (3H, m), 6.71 (2H, d, J = 8.4), 6.77
(2H, d, J = 8.6), 6.95 (2H, d, J = 8.4), 7.01 (2H, d, J = 8.4).
The 1H-NMR spectrum was consistent with published data.4,23,24
All other commercial reagents were obtained from Nacalaitesque
Co., Ltd. or Wako Pure Chemicals Co., Ltd., unless otherwise
noted.
Experimental
Plant materials
A cell-suspension culture of A. officinalis cv. Akuseru was initiated
and elicited with autoclaved Fusarium solani IFO4542 mycelia for
18 h as described previously.8 The cells were collected using a
strainer and washed with distilled water, and the excess water was
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Isolation of ZHR from A. officinalis
(+)-enantiomer (tR = 11.2–11.4 min: from the top to the end of
the second peak) were recovered. The solvent was evaporated off.
Each enantiomer thus obtained was mixed with 0.1 mg of acetosy-
ringone, and subjected to GC-MS measurement. Enantiomeric
compositions of THRs were estimated by using the amounts of
each enantiomer quantified by comparing chromatographic peak
areas of molecular ions between THR and acetosyringone.
We isolated ZHR from A. officinalis cells as described
previously.8,25 Freeze-dried A. officinalis cells were extracted with
hot methanol. After b-glucosidase treatment, the extracts were pu-
rified with silica gel TLC and reversed-phase HPLC using a Waters
Novapak C18 column, giving rise to ZHR: m/z [EI, ZHR TMS
ether] 396 (M+, 100%), 381 (27), 230 (91), 217 (47) and 179 (39).
Conclusion
Isolation of EHR from C. japonica
We have demonstrated that the combination of A. officinalis HRS
subunits controls enantioselectivity as well as cis/trans isomerism
in hinokiresinol formation.
Freeze-dried C. japonica cells were extracted with hot methanol
and then treated with b-glucosidase. The extracts were purified
using silica gel TLC and reversed-phase HPLC using a Waters
Novapak C18 column, yielding EHR: m/z [EI, EHR TMS ether]
396 (M+, 100%), 381 (25), 230 (89), 217 (36) and 179 (31).
Acknowledgements
We thank Professor Hideo Ohashi, Gifu University, for the
authentic (E)-hinokiresinol from heartwood of C. obtusa. We also
thank Miyuki Nakamura for help with the maintenance of A.
officinalis and C. japonica cells.
Preparation of ZHR with the A. officinalis enzyme
Enzyme preparation; a crude enzyme preparation from A. offic-
inalis cells8 was incubated with 4-coumaryl 4-coumarate (final
conc. 0.6 mM) as a substrate at 30 ◦C for 3 h as described
previously.10 The reaction mixture was then extracted with
three times EtOAc, the organic layer was then washed with
saturated aqueous NaCl, dried over Na2SO4, and concentrated in
vacuo. ZHR was isolated and purified by TLC and reversed-phase
HPLC using a Waters Novapak C18 column.
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coumaryl 4-coumarate as described previously.10
Thus, the mixture containing equal amounts of recHRSa and
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recHRSa was incubated with the same substrate at 30 ◦C for 1 h.
The reaction mixture was extracted three times with EtOAc. The
organic layer was washed with saturated aqueous NaCl, dried over
Na2SO4, and concentrated in vacuo. The reaction was repeated
twice, and the products were combined and purified to afford
EHR. Incubation of the same substrate with recHRSb giving rise
to EHR was carried out exactly as described above.
Determination of enantiomer excess of hinokiresinols
Isolated and enzymatically prepared hinokiresinols were individ-
ually hydrogenated to THR. Each of the obtained THRs was
subjected to the chiral HPLC analysis, and the enantiomeric
composition of THR was determined by calculating the peak area
of each enantiomer recorded by the UV detector. In addition, the
enantiomeric compositions of THRs were confirmed by GC-MS
analysis. To avoid contamination by antipodes, the former half of
the peak of (-)-enantiomer (tR = 10.6–10.8 min: from the start
to the top of the first peak) and the letter half of the peak of
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