Stereochemistry in the reduction of enones by the reductase from Euglena gracilis Z

Article abstract of DOI:10.1016/S0031-9422(97)00858-3

A reductase, which catalyses the NADH-dependent reduction of the C=C bond adjacent to the carbonyl group, was characterized with regard to the stereochemistry of the hydrogen transfer into the substrate. The reductase was isolated from Euglena gracilis Z and was found to reduce stereospecifically the C=C bond of carvone by anti-addition of hydrogen from the si face at α-position to the carbonyl group and the re face at β- position. The hydrogen atoms participating in the enzymatic reduction at α- and β-position to the carbonyl group originate from the medium and the pro- 4R hydrogen of NADH, respectively.

Full text of DOI:10.1016/S0031-9422(97)00858-3

Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 38Ð42\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
Pergamon
\
9920Ð8311:87:,Ðsee front matter
PII] S9920Ð8311"86#99747Ð2
STEREOCHEMISTRY IN THE REDUCTION OF ENONES BY THE
REDUCTASE FROM EUGLENA GRACILIS Z
KEI SHIMODA\ TOSHIFUMI HIRATAꢀ and YOSHIAKI NOMA$
Department of Chemistry\ Faculty of Science\ Hiroshima University\0!2!0 Kagamiyama\ Higashi!Hiroshima 628!7415\
Japan^ $ Faculty of Domestic Science\ Tokushima Bunri University\ Yamashiro!cho\ Tokushima 669!7403\ Japan
"Received in revised form 7 September 0886#
Key Word Index*Eu`lena `racilis Z^ Euglenaceae^ stereochemistry^ enone reductase^ carvone^
verbenone^ pulegone^ piperitenone[
Abstract*A reductase\ which catalyses the NADH!dependent reduction of the C1C bond adjacent to the
carbonyl group\ was characterized with regard to the stereochemistry of the hydrogen transfer into the
substrate[ The reductase was isolated from Eu`lena `racilis Z and was found to reduce stereospeci_cally the
C1C bond of carvone by anti!addition of hydrogen from the si face at a!position to the carbonyl group and
the re face at b!position[ The hydrogen atoms participating in the enzymatic reduction at a! and b!position to
the carbonyl group originate from the medium and the pro!3R hydrogen of NADH\ respectively[ Þ 0887
Elsevier Science Ltd[ All rights reserved
INTRODUCTION
subjected to a Blue Toyopearl column to give a pure
enzyme preparation "Table 0#[ The puri_ed enzyme
preparation was homogeneous as judged by the pres!
ence of a single protein band on the SDS gel elec!
trophoresis\ as shown in Fig[ 0[
The Mr of the enzyme was estimated as about 44 k
by gel _ltration on a Sephadex G!049 column[ The
SDS gel electrophoresis of the enzyme preparation
showed a single band at about 44 k "Fig[0#[ These
observations indicate that the native enzyme is mono!
mer of 44 k protein[ The enzyme shows a pH optimum
at 6[3 with half maximal activity at 5[2 and 7[0 in 2!
morpholinopropanesulfonic acid!NaOH bu}er[ The
enzyme required NADH and NADPH as coenzymes\
and the activity in presence of NADH was twice as
high as that in presence of NADPH[ FAD and FMN
were ine}ective for the enzyme activity[
The substrate speci_cities of the enzyme were inves!
tigated by use of several enones as the substrates\ as
shown in Table 1[ The data show a rather limited
substrate speci_city^ only the C1C bond having a
hydrogen atom at the position b to the carbonyl group
can be reduced[ When carvone "0# was used as the
substrate\ the "R#!enantiomer "0a# was reduced enanti!
oselectively to yield "0R\ 3R#!dihydrocarvone "2#\ and
the conversion was higher than that of "S#!enantiomer
Hitherto known biochemical reduction of the C1C
bond of a\b!unsaturated ketones were most inves!
tigated with microorganisms ð0Ð2Ł and animals ð3\ 4Ł[
Recently\ three kinds of enone reductases were iso!
lated from the cultured cells of Nicotiana tabacum
and the stereochemical studies in the reduction of the
C1C bond by these reductases have been reported
ð5Ð7Ł[ On the other hand\ Noma et al[ reported that
the cultured cells of Eu`lena `racilis Z classi_ed in
Phytomastigophoria also reduce the C1C bond of
carvone "0# with high stereospeci_city ð8\ 09Ł[ In con!
nection with the studies on the enone reductase from
plant cells\ we now investigate the isolation and
characterization of the enone reductase from E[ `ra!
cilis and the stereochemistry in the reduction of the
C1C bond of enones with the reductase[
RESULTS AND DISCUSSION
A microsomal enzyme was obtained from the E[
racilis cell cultures by extraction with Triton X!099
`
and then subjected to chromatography on a Sephadex
G!14 column and a diethylaminoethyl "DEAE# Toyo!
pearl column[ The activity of the enzyme was assayed
at pH 6[3 using "R#!carvone "0a# as the substrate in the
presence of NADH[ The active fraction was further
"
0b#\ yielding "0R\ 3S#!isodihydrocarvone "3#[ No
reduction of the enones such as 2!methyl!
cyclohexenone\ "0S\ 4S#!verbenone "1a#\ "0R\ 4R#!ver!
benone "1b#\ "R#!pulegone "4# and piperitenone "5#
ꢀ
Author to whom correspondence should be addressed[ was observed[ These observations show that the
E!mail] thirataÝsci[hiroshima!u[ac[jp
enzyme is similar to the carvone reductase isolated
3
8

4
9
K[ SHIMODA et al[
Table 0[ Puri_cation of the reductase from E[ `racilis Z
Total protein
mg
Total activity
unit×09
Sp[ act[
units per g protein
3
Fold
Crude extract
DEAE!Toyopearl
AF!Blue Toyopearl
014
6
9[0
1[1
0[4
9[92
0[6
10
29
0
01
07
O
O
O
2a
1b
1a
O
O
O
2b
3
4
O
O
5
6
Fig[ 0[ SDS!gel electrophoretic analyses of the reductase
from E[ `racilis cell cultures[ "a# DEAE Toyopearl fraction\
"b# Blue Toyopearl fraction[
deuterium in the resultant deuterated product was
examined[ The labelling pattern of deuterium and the
from the cultured cells of N[ tabacum ð5\ 6Ł in respect deuterium contents in the resulting dihydrocarvone
of substrate speci_city[ "2# were determined by NMR and mass spectroscopy[
The fact that "0R\ 3R#!dihydrocarvone "2# and "0R\ Dihydrocarvone "2# produced in the incubation in the
1
3
S#!isodihydrocarvone "3# were produced by the enzy! presence of NADH in H O ðexperiment "b#Ł showed
1
matic reduction of "R#! and "S#!carvone "0a and 0b#\ a peak at m:z 042 in the mass spectrum\ i[e[ one mass
respectively\ indicates that the hydrogen attack at the unit higher than the molecular ion peak "m:z 041# of
conjugated C1C bond takes place stereospeci_cally dihydrocarvone "2# produced in the control experi!
from the si face at C!0[ However\ the stereochemistry ment "a#[ This indicates that the deuterium atom orig!
1
of the hydrogen attack at C!5 of carvone "0# was inating from H O is incorporated into dihydro!
1
unknown[ To complete the stereochemical studies on carvone[ On the other hand\ the mass spectrum
the reduction of the C1C bond with the enzyme prep! of dihydrocarvone "2#\ produced when "3R#!
1
aration\ we investigated the stereochemistry of the ð3− HŁNADH was included in the incubation mix!
hydrogen attack to 0a by performing the following ture ðexperiment "c#Ł\ showed a peak at m:z 042
¦
1
four experiments[ "R#!Carvone "0a# was incubated ðM¦0Ł [ However\ when the "3R#!ð3− HŁNADH
1
with the enzyme preparation in the presence of] "a# was replaced by "3S#!ð3− HŁNADH ðexperiment "d#Ł\
NADH in H O^ "b# NADH in H O^ "c# "3R#! no deuteration of dihydrocarvone "2# was observed[
ð3− HŁNADH in H O and "d# "3S#!ð3− HŁNADH in These observations indicate that only the pro!3R
1
1
1
1
1
1
H O[ Through these experiments\ the orientation of hydrogen of NADH is incorporated into dihy!
1

Reduction of enones by reductase from E[ `racilis Z
40
Table 1[ Substrate speci_city in the reduction of various enones with the reductase from E[
`
racilis Z
"a#
Substrate
Conversion:)
Product
"
"
"
"
"
R#!Carvone "0a#
S#!Carvone "0b#
0S\4S#!Verbenone "1a#
0R\4R#!Verbenone "1b#
R#!Pulegone "4#
12
5
9
9
9
"0R\3R#!Dihydrocarvone "2#
"0R\3S#!Isodihydrocarvone "3#
*
*
*
Cyclohex!1!en!l!one
34
4
9
Cyclohexanone
"R#!1!Methylcyclohexan!l!one
*
*
1
2
!Methylcyclohex!1!en!l!one
!Methylcyclohex!1!en!l!one
Piperitenone "5#
9
"a# Conversions are expressed as the percentage of the products obtained in the reduction
of summarized substrates under the assay conditions[
drocarvone "2# during the enzymatic reduction of the
double bond[ The labelled sites in the deuterium!lab!
1
elled dihydrocarvone were determined from H NMR
spectroscopy[ The spectrum of dihydrocarvone "2#
1
produced in the presence of NADH in H O showed
1
1
1
only a signal at d 1[26 due to the H at C!0[ The H!
enrichment factors at the labelled site was determined
1
from the intensity of the corresponding H peak on
1
the basis of the peak intensity of natural H in CHCl2
used as the solvent and was 88)[ However\ dihy!
drocarvone produced in the presence of "3R#!
1
ð3− HŁNADH exhibited a signal at d 0[26 "88)
1
enrichment# due to the H at C!5 trans!oriented to the
hydrogen atom at C!0[ These observations indicate
that the deuterium atoms at C!5 and C!0 of the deut!
erated dihydrocarvone originate from "3R#!
1
1
ð3− HŁNADH and H O\ respectively[
1
Thus\ it was concluded that the reduction of the
C1C bond of enones with the reductase from E[
`
racilis occurs stereospeci_cally by the anti!addition
of hydrogen atoms from the si face at C!0 and the re
face at C!5 of the C1C bond of carvone\ as shown
in Fig[2[ The hydrogen atoms participating in the
reduction at a! and b! positions of the carbonyl group
originate from the medium and the pro!3R hydrogen
of NADH\ respectively[ The reductase isolated from
the cultured cells of E[ `racilis is similar to the carvone
reductase from N[ tabacum in respect of stereo!
chemistry of the hydrogen transfer\ but composed of
di}erent Mr from the carvone reductase\ i[e[\ although
the reductase from E[ `racilis is a monomer of 44 k
protein\ the carvone reductase from N[ tabacum is
composed of two identical subunits of a 11 k protein^
although the carvone reductase had been reported to
be a tetramer of 11 k protein by Tang et al[ ð6Ł\ our
recent investigation showed the reductase to be dimer
of 11 k protein[
Fig[ 1[ NMR spectra of dihydrocarvone "2# obtained by
0
the enzymatic reduction of "R#!carvone "0a#] "a# H NMR
EXPERIMENTAL
spectrum for the product by the incubation with NADH in
1
H
1
O^ "b# H NMR spectra for the products by the incubation
Analytical and preparation
1
with NADH in
ð3− HŁNADH in H
H
1
O^ "c# the incubation with "3R#!
O^ and "d# incubation with "3S#!
1
TLC] 9[14 mm silica gel "Merck silica gel 59^ GF143#[
GC] FID and a glass column "2 mm×1 m# packed
1
1
1
ð3− HŁNADH in H O[

4
1
K[ SHIMODA et al[
HS
HR
N-R
NADH
CONH2
re
O
O
H
anti–addition
O
si
H
1a
3
Fig[ 2[ Stereochemistry in the reduction of "R#!carvone by the reductase from Eu`lena `racilis Z[
1
with 04) DEGS at 099> or a capillary column "9[14 Preparation of "3S#!ð3− HŁNADH
mm×14 m# coated with 9[14 mm CP!cyclodextrin b
Following the reported method ð02Ł\ "3S#!
1
25M!08[ GC!MS] a GCMS!QP 1999A spectrometer
1
ð3− HŁNADH was prepared by enzymatic reduction
0
1
by El mode at 69 eV[ H NMR] 169 MHz in H O and
CDCl with Me Si as internal standard[ H NMR]
1
1
¦
of ð3− HŁNAD "019 mg# with EtOH "399 mg# and
yeast alcohol dehydrogenase "16 units^ 09 mg#[ The
reaction mixture was chromatographed on a DEAE!
1
2
3
3
0[4 MHz in CHCl by use of the solvent peak as an
2
internal standard[
1
Toyopearl column to give "3S#!ð3− HŁNADH "29
1
0
mg\ 88) H!enrichment#\ H NMR "D O# d 1[70 "0H\
1
brs\ 3R!H#\ 5[08 "0H\ d\ OCH # and 5[81 "0H\ s\ 1!H#[
1
Substrate
1
4
"
0S\4S#!"−#!Verbenone\ ðaŁ −085 "c 9[38\ EtOH#
D
14
and "0R\4R#!"¦#!verbenone\ ðaŁ
¦100 "c 0[4\ Puri_cation of enzyme
D
CHCl # were prepared from "−#!a!pinene and "¦#!a!
2
Cultured cells of E[ `racilis Z were prepared as
described in Ref[ ð03Ł^ cells cultured for 2 weeks were
used in the present work[ All puri_cation procedures
were carried out at 3>[ Cultured cells "4 g# were frozen
pinene\ respectively\ by oxidation with t!butyl chro!
1
4
mate ð00Ł[ "R#!"−#!Carvone\ ðaŁ −59[0 "neat# was
D
1
4
purchased from Aldrich[ "S#!"¦#!Carvone\ ðaŁ
D
14
¦
46[0 "neat# and "R#!"¦#!pulegone\ ðaŁ ¦11[2 "c
D
with liq[ N \ ground and homogenized with 099 mM
1
3
[5\ EtOH# were donated by Takasago Perfumery Co[
Ltd[ 1!Metylcyclohex!1!en!l!one was prepared from
!methylcyclohexan!l!one by chlorination with sul!
Na!Pi bu}er "pH 5[7\ 09 ml# containing 9[14 M
sucrose\ 09) glycerol\ 4 mM dithiothreitol\ 4 mM
Na S O and 9[4) Triton X!099 in a Waring Blender[
1
1
1
4
furyl chloride followed by dehydrochlorination with
collidine according to the reported method ð01Ł[
Cyclohex!1!en!l!one and 2!methylcyclohex!1!en!l!one
The homogenate was _ltered through cheesecloth\ and
the _ltrate was centrifuged at 09\999 ` for 09 min[ The
supernatant was desalted through a Sephadex G!14
column previously equilibrated with Tris bu}er "ph
¦
were commercial products[ NADH\ NADP \
NADPH\ FAD and FMN were purchased from
Oriental Yeast Co[ Ltd[^ Sephadex G!14 was from
7
[9# containing 0 mM Na S O and 0 mM dithi!
1 1 4
othreitol[ The protein fraction was subsequently
loaded onto a DEAE!Toyopearl column "2[0×12 cm#
equilibrated with the standard bu}er[ After washing
with the bu}er soln\ the enzymes were eluted with a
liner gradient of NaCl "9½9[4 M in the standard
bu}er#[ The active enzyme fractions were dialysed
against the standard bu}er[ The enzyme soln was sub!
Pharmacia^ DEAE Toyopearl and AF!Blue Toyo!
1
pearl 549ML were from TOSOH Co[ Ltd[^ H O
1
"88[8) atom D# was from Aldrich[
1
Preparation of "3R#!ð3− HŁNADH
Following the reported method ð02Ł\ "3R#! sequently subjected to further puri_cation on an AF!
1
ð3− HŁNADH was prepared by reduction of b! Blue Toyopearl column "0[1×8 cm# equilibrated with
¦
NAD "019 mg# with EtOH!d "88) enrichment^ 399 the standard bu}er[ After washing with the bu}er
5
mg# and yeast alcohol dehydrogenase "16 units^ 09 soln\ the enzymes were eluted with a linear NaCl
mg#[ The crude product was subjected to chro! gradient "9½1 M in the standard bu}er#[ E/uent
matography on a DEAE Toyopearl column to give fractions containing the active enzyme were pooled
1
1
0
"
3R#!ð3− HŁNADH "59 mg\ 88) H!enrichment#\ H and used for experiments[
NMR "D O# d 1[55 "0H\ brs\ 3S!H#\ 5[07 "0H\ d\
The Mr values of the reductase were estimated by
1
OCH # and 5[89 "0H\ s\ 1!H#[
gel _ltration through a Sephadex G!049 column
1

Reduction of enones by reductase from E[ `racilis Z
42
¦
1
"
0[4×64 cm#\ using aldolase\ bovine serum albumin\ "M \ 8#\ 026 "8#\ 098 "11#\ 84 "40# and 56 "099#^ H
1
ovalbumin and ribonuclease A as protein markers[ NMR "CHCl # no H!signal was detected[
2
According to method of Ref[ ð04Ł\ 9[3) SDS!04)
PAGE of the enzyme was preformed[ The Mr of
enzyme was determined by reference to the mobilities
of proteins of known Mr "LMW electrophoresis cali!
Incubation of "R#!carvone "0a# with the enzyme
1
preparation in the presence of H O
1
bration kit from Pharmacia#[
"
R#!carvone "29 mmol# was incubated with the
1
enzyme fraction in the bu}er prepared with H O to
give H!labelled dihydrocarvone] m:z "rel[ int[# 042
M¦0 \ 4#\ 027 "4#\ 009 "05#\ 84 "23# and 56 "099#^ H
NMR "CHCl # d 1[26 "s\ 0− H#[
1
1
Optimum pH
¦
1
"
The reaction mixture was composed of 899 ml 099
mM 2!"N!morpholino#propane sulfonic acid bu}er
with pH adjusted from 5[9 to 7[4\ 2 mmol "R#!carvone\
1
2
Acknowled`ements*The authors thank the Instru!
ment Center for Chemical Analysis of Hiroshima Uni!
5
mmol NADH\ 9[0 ml 0) Triton X!099 and 9[0 ml
enzyme prepn[ The enzyme activity was determined
according to the same method as the standard assay[
The standard enzyme assay mixture contained 0 ml
enzyme prepn in 14 mM Na!Pi bu}er with pH
adjusted to 6[3\ 2 mmol "R#!carvone\ 5 mmol NADH
and 9[0 ml 0) Triton X!099[ The mixture was incu!
0
1
versity for the measurements of H and H NMR
spectra\ and also thank Professor Katsuo Ohkata of
Hiroshima University for permission to use a Shi!
madzu QP!1999A GC!MS spectrometer[ The authors
wish to express their gratitude to Emeritus Professor
Shozaburo Kitaoka\ University of Osaka Prefecture\
for supplying the culture of Eu`lena `racilis Z[
bated for 01 h at 25> and then extracted with Et O and
1
subjected to GC and GC!MS analyses[ The enzyme
activity was expressed as the amount of dihy!
drocarvone produced[
REFERENCES
0
1
2
3
4
[ Davidson\ S[ J[\ Methods Enzymolo`y\ 0858\ 04\
45[
[ Miller\ T[ L[ and Hessler\ E[ J[\ Biochimica et
Biophysica Acta\ 0869\ 191\ 243[
[ Kergomard\ A[\ Renard\ M[ F[ and Veshambre\
H[\ Journal of Or`anic Chemistry\ 0871\ 36\ 681[
[ Berseus\ O[\ Danielsson\ H[ and Kallner\ A[\ The
Journal of Biolo`ical Chemistry\ 0854\ 139\ 1285[
[ Berseus\ O[ and Bjorkhem\ I[\ European Journal
of Biochemistry\ 0856\ 1\ 492[
Incubation of enones with the enzyme preparation
5
Following the standard assay method\ enzymatic
reduction of several enones was preformed[ The
reduction products were identi_ed with NMR\ GC!
MS and CD spectroscopy[ Enantiomeric purity was
determined from the peak area of GLC with CP cyclo!
dextrin b 125M!08 column[ In the case of "R#!carvone
as substrate\ the product was "0R\ 3R#!dihydro!
¦
carvone ] m:z "rel[ int[# 041 "M \ 05)#\ 026 "09#\ 098
0
5[ Hirata\ T[\ Tang\ Y[\ Okano\ K[ and Suga\ T[\
"
00#\ 84 "65# and 56 "099#^ H NMR "CDCl # d 0[91
2
Phytochemistry\ 0878\ 17\ 2220[
"2H\ d\ J ꢁ 5[4 Hz\ 0!Me#\ 0[26 "0H\ qd\ J ꢁ 01[7 and
6
7
[ Tang\ Y[ and Suga\ T[\ Phytochemistry\ 0881\ 20\
488[
[ Shimoda\ K[\ Ito\ D[I[\ Izumi\ S[ and Hirata\ T[\
Journal of the Chemical Society\ Perkin Trans!
action 0\ 0885\ 244[
2
7
[7 Hz\ 5!H trans!oriented to the 0!H#\ 0[63 "2H\ s\
!Me#\ 1[02 "0H\ dq\ J ꢁ 01[7 and 1[7 Hz\ 5!H cis!
1
oriented to the 0!H# respectively[ Incubation of 1!
methylcylohexen!l!one with the reductase produced
"R#!1!methylcyclohexan!l!one " × 88) enantiomeric
8
[ Noma Y[ and Asakawa\ Y[\ Phytochemistry\
excess#] CD ðuŁmax −889 at 178 nm "c 9[91\ MeOH#
ðRef[ ð05Ł] ðuŁmax −876 at 177 nmŁ
0
881\ 20\ 1998[
0
9[ Noma Y[ and Asakawa\ Y[\ Biotechnolo`y in
A`riculture and Forestry\ in Vol[ 30[ "Medical and
Aromatic Plants#\ ed[ Y[ P[ S[ Bajaj\ Springer
Verlag\ Berlin\ 0886 "in press#[
Incubation of "R#!carvone "0a# with the enzyme
1
preparation in the presence of "3R#!ð3− HŁNADH
0
0[ Matsuura\ T[ and Fujita\ K[\ Journal of Science
of the Hiroshima University Ser[ A\ 0841\ 05\ 062[
1[ Warnho}\ E[ W[\ Martinx\ D[ G[ and Johnson\
W[ S[\ Or`anic Synthesis Collective\ Vol[ 3[ 0852\
p[ 051[
2[ Colowick\ S[ P[ and Kaplan\ N[ O[\ Methods
Enzymolo`y\ 0846\ 3\ 739[
3[ Noma Y[\ Takahashi\ H[ and Asakawa\ Y[\
Phytochemistry\ 0880\ 29\ 0036[
The enzymatic reduction of "R#!carvone "29 mmol#
with the labelled NADH "59 mmol# was performed
1
0
in the same procedure as above to give H!labelled
¦
dihydrocarvone] m:z "rel[ int[# 042 "M¦0 \ 3#\ 027
1
"
3#\ 009 "06#\ 84 "34# and 56 "099#^ H NMR "CHCl #
2
1
0
0
0
d 0[26 "s\ 5! H trans!oriented to the 0!H#[
Incubation of "R#!carvone "0a# with the enzyme
1
preparation in the presence of "3S#!ð3− HŁNADH
4[ Laemmli\ U[ K[\ Nature "London#\ 0869\ 166\ 579[
1
In the presence of "3S#!ð3− HŁNADH\ "R!carvone 05[ Cheer\ C[ J[ and Djerassi\ C[\ Tetrahedron Letters\
was converted to dihydrocarvone] m:z "rel[ int[# 041
0865\ 32\ 2766[