Structure-based insight into the asymmetric bioreduction of the C=C double bond of α,β-unsaturated nitroalkenes by pentaerythritol tetranitrate reductase

Article abstract of DOI:10.1002/adsc.200800561

Biocatalytic reduction of α- or β-alkyl-barylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene (1) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes (4a-d) to their equivalent (S)-nitropropanes 9a-d. The enzyme shows a preference for the (Z)-isomer of substrates 4a-d, providing almost pure enantiomeric products 9a-d (ees up to > 99%) in quantitative yield, whereas the respective (E)-isomers are reduced with lower enantioselectivity (63-89% ee) and lower product yields. 1-Aryl-2-nitropropenes (5a, b) are also reduced efficiently, but the products (R)-10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene (1) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the (E)- and (Z)- substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase.

Full text of DOI:10.1002/adsc.200800561

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DOI: 10.1002/adsc.200800561
Structure-Based Insight into the Asymmetric Bioreduction
of the C=C Double Bond of a,b-Unsaturated Nitroalkenes by
Pentaerythritol Tetranitrate Reductase
Helen S. Toogood,a,d Anna Fryszkowska,b,d Victoria Hare,a Karl Fisher,c
Anna Roujeinikova,a David Leys,a John M. Gardiner,b Gill M. Stephens,c
and Nigel S. Scruttona,*
a
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street,
Manchester M1 7DN, U.K.
Fax : (+44)-161-306-8918; phone: (+44)-161-306-5152; e-mail: nigel.scrutton@manchester.ac.uk
Manchester Interdisciplinary Biocentre, Department of Chemistry, University of Manchester, 131 Princess Street,
b
Manchester M1 7DN, U.K.
c
Manchester Interdisciplinary Biocentre, School of Chemical Engineering and Analytical Sciences, University of
Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
The first two authors contributed equally to this work.
d
Received: September 12, 2008; Published online: November 19, 2008
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.200800561.
Abstract: Biocatalytic reduction of a- or b-alkyl-b- are also reduced efficiently, but the products (R)-10
arylnitroalkenes provides a convenient and efficient have lower optical purities. The structure of the
method to prepare chiral substituted nitroalkanes. enzyme complex with 1-nitrocyclohexene (1) was de-
Pentaerythritol tetranitrate reductase (PETN reduc- termined by X-ray crystallography, revealing two
tase) from Enterobacter cloacae st. PB2 catalyses the substrate-binding modes, with only one compatible
reduction of nitroolefins such as 1-nitrocyclohexene with hydride transfer. Models of nitropropenes 4 and
(1) with steady state and rapid reaction kinetics com- 5 in the active site of PETN reductase predicted that
parable to other old yellow enzyme homologues. the enantioselectivity of the reaction was dependent
Furthermore, it reduces 2-aryl-1-nitropropenes (4a– on the orientation of binding of the (E)- and (Z)-
d) to their equivalent (S)-nitropropanes 9a–d. The substrates. This work provides a structural basis for
enzyme shows a preference for the (Z)-isomer of understanding the mechanism of asymmetric biore-
substrates 4a–d, providing almost pure enantiomeric duction of nitroalkenes by PETN reductase.
products 9a–d (ees up to>99%) in quantitative
yield, whereas the respective (E)-isomers are re- Keywords: asymmetric hydrogenation; biocatalysis;
duced with lower enantioselectivity (63–89% ee) and bioreduction; nitroalkenes; pentaerythritol tetrani-
lower product yields. 1-Aryl-2-nitropropenes (5a, b) trate reductase; stereocontrol
Introduction
or asymmetric transfer hydrogenation of olefins using
a Jacobsen-type organocatalyst.[5] Asymmetric reduc-
Nitroalkanes are versatile, inexpensive synthons that tion is especially attractive as up to two stereogenic
can be converted readily to the corresponding amines, centres can be formed. Therefore, the asymmetric re-
aldehydes, carboxylic acids, oximes, hydroxylamines, duction of activated C=C bonds by microorganisms or
or denitrated compounds.[1] Enantiomerically pure ni- enzymes has recently received a lot of interest since
troalkanes have been produced by a variety of chemi- the catalysts are environmentally benign, the reac-
cal methods. These include asymmetric conjugate ad- tions can be performed under mild conditions, sub-
dition to nitroalkenes,[2] asymmetric conjugate addi- strate ranges are broad, and product enantiopurity is
tion of nitroalkanes to various a,b-unsaturated bonds generally excellent.[6]
(C=C or C=N),[3] asymmetric conjugate reduction of
nitroalkenes using chiral transition metal catalysis,[4] family have great potential in industrial biocatalysis
Enzymes from the old yellow enzyme (OYE)
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Scheme 1. (a) General mechanism of the biocatalytic reduction of a,b-unsaturated alkenes catalyzed by the OYE family.
(b) General mechanism of the biocatalytic reduction of a,b-unsaturated nitroalkenes involving nitronate formation and aci-
nitro tautomerisation of the product formed. EWG=electron withdrawing group; *=chiral centre.
as they catalyse a range of synthetically useful reduc- the reduction of explosives, including nitroaromatic
tions.[6–11] These FMN-containing enzymes catalyse compounds (e.g., trinitrotoluene, TNT), and cyclic tri-
the NAD(P)H-dependent reduction of activated a,b- azines (e.g., Royal Demolition Explosive).[18,19] The
unsaturated alkenes (Scheme 1).[6] Asymmetric biore- enzyme also cleaves nitrate esters, such as pentaery-
duction by OYEs proceeds in a step-wise fashion with thritol tetranitrate (PETN) and the vasodilator nitro-
a mechanism resembling Michael-type conjugate ad- glycerin.[18–20]
dition of hydride to activated alkenes[12] with the addi-
Owing to its broad specificity, we conjectured that
tion of [H2] proceeding in a trans-fashion,[12] although PETN reductase might be an excellent candidate for
there is evidence for cis-addition in other related industrial biocatalysis. This enzyme is extremely
classes of enzymes.[6,13]
stable, and can be expressed to high levels in Escheri-
Recently, the asymmetric bioreduction of a variety chia coli.[21] It has been characterised extensively at
of a,b-unsaturated aldehydes, ketones, maleimides both the mechanistic and structural level with sub-
and nitroalkenes was demonstrated with various strates such as 2-cyclohexenone and nitroaromatic ex-
members of the OYE family.[6–11] Highly enantioselec- plosives.[18] Multiple structures of PETN reductase
tive reduction of b,b-disubstituted nitroalkenes has have been determined to high resolution,[22] both in
been shown to occur in organisms such as bakerꢁs the presence and absence of bound substrates and in-
yeast,[14] and using OYE homologues from Lycopersi- hibitors.[18,22–25] This enabled us to take a structure-
con esculentum (OPR1 and OPR3),[8,10] bakerꢁs yeast driven approach to aid in the prediction of both sub-
Saccharomyces carlsbergensis (OYE1-3),[9] Zymomo- strate selectivity and the stereochemical outcome of
nas mobilis (NCR),[9] and Bacillus subtilis (YqjM)[8] the reactions. In this paper, we have explored the
and a new reductase from Clostridium sporogenes.[15] ability of PETN reductase to catalyse the enantiose-
The enzymes exhibited different substrate selectivi- lective reduction of a variety of both (E)- and (Z)-a/
ties, but, in general, the reactions were catalysed with b-alkyl-b-arylnitroalkenes to examine its potential as
high enantioselectivity and yield. In contrast, biologi- a generic biocatalyst.
cal reduction of a,b-disubstituted nitroalkenes pro-
ceeded with poor enantioselectivity (<50% ee).[15–17]
Some of the OYE family members are known to Results and Discussion
catalyse a broad range of chemical reactions. For ex-
ample, pentaerythritol tetranitrate reductase (PETN Kinetic Analysis of Nitroalkene Reduction
reductase) from Enterobacter cloacae st. PB2 not only
acts as an ene reductase (e.g., for reduction of 2-cy- To explore its reactivity with nitroalkenes, PETN re-
clohexenone and some steroids),[18] but also catalyses ductase was initially tested for reduction of the com-
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Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond
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biocatalysis (Table 1a; Supporting Information Fig-
ure S1). PETN reductase had 5- to 10-fold higher Km
values for the compounds tested compared to OYE1
(Table 1a), making the specificity of the reaction (kcat/
Km) between 11- and 45-fold lower for PETN reduc-
tase. Nevertheless, the Km values were still <1 mM,
and, therefore, would not place restrictions on indus-
trial exploitation.
A study of the reoxidation of PETN reductase by
1-nitrocyclohexene using stopped-flow methods
showed a hyperbolic dependence of the rate constant
with substrate concentration, as opposed to OYE1
where only a second order rate constant was deter-
mined.[27] In contrast, only second order rate constants
could be calculated for the oxidative half reaction
with nitroalkenes 2–3 due to the low solubility of the
substrates and relatively high Kd values compared to
OYE1 (Table 1b; Supporting Information Figure S1).
To investigate the biocatalytic potential of PETN
reductase we monitored the reduction of a variety of
nitroolefins (E)-1–3 and (E)- and (Z)-4–5 to complete
substrate depletion under both homogeneous (maxi-
mum 5% ethanol in water) and biphasic conditions.
Scheme 2. Substrates and products of C=C double bond bio-
reduction catalysed by PETN reductase.
mercially available substrates 1-nitrocyclohexene (1), Initially the reactions were monitored spectrophoto-
(E)-1-nitro-2-phenylethene (2) and (E)-1-nitro-2-(2’- metrically under homogeneous conditions using a glu-
thienyl)-ethene (3) (Scheme 2).
cose 6-phosphate dehydrogenase-NADP+/NADPH
The steady-state kinetic parameters were deter- cofactor regeneration system to avoid the problem of
mined using the stopped-flow method of Gibson et NADPH spectrum overlap with the substrates.[27] All
al.[26] monitoring the reduction of the flavin coenzyme reactions were performed anaerobically to avoid
directly (~450 nm) to avoid the interference from the enzyme inactivation by reactive oxygen species, and
overlapping spectra of NADPH and the substrate at to eliminate the competition between the oxidation of
shorter wavelengths. The catalytic rate for reduction the flavin cofactor by the nitroalkene substrates and
of 1-nitrocyclohexene was twice as fast with PETN re- oxygen.
ductase as with OYE1,[27] whilst the PETN reductase-
The spectral changes due to the reduction of (E)-
catalysed reduction of nitroalkenes (E)-2 and (E)-3 and (Z)-(4’-chlorophenyl)-nitropropenes under homo-
was 4–5 times as rapid, suggesting that PETN reduc- geneous conditions were monitored continuously until
tase would be an excellent candidate for industrial reaction completion (data for substrates 4c and 5b are
Table 1. (a) Steady state kinetic constants and (b) rate constants for the oxidative half-reaction of PETN reductase with
three nitro olefin substrates. In the case of (E)-2–3, kox/Kd was determined from the initial slope of the kobs vs. substrate con-
centration plot where the substrate concentration is well below Km.
(a) Steady State
Compound
PETN reductase
Km [mM]
OYE1[a]
Km [mM]
kcat [sꢀ1]
kcat/Km [sꢀ1 Mꢀ1]
kcat [sꢀ1]
kcat/Km [sꢀ1 Mꢀ1]
1
9.5ꢁ1.0
20.9ꢁ0.8
21.4ꢁ1.0
971ꢁ146
341ꢁ42
413ꢁ39
9773
61290
51815
4.4ꢁ0.2
5.0ꢁ0.1
4.3ꢁ0.2
10ꢁ1
440,000
1,190,476
558,441
(E)-2
(E)-3
4.2ꢁ0.3
7.7ꢁ0.5
(b) Oxidative Half Reaction
Compound
PETN reductase
Kd [mM]
OYE1[a]
Kd [mM]
kox [sꢀ1]
kox/Kd [sꢀ1 Mꢀ1]
kox [sꢀ1]
kox/Kd [sꢀ1 Mꢀ1]
1
41.7ꢁ2.1
ND
ND
6.4ꢁ0.7
>3 mM
>2 mM
6502
4250
1272
–
–
610,000
2,800,000
1,000,000
(E)-2
(E)-3
420ꢁ20
0.15ꢁ0.02
170ꢁ30
0.17ꢁ0.03
[a]
Data from[27]; ND=not determined.
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Figure 1. Spectral changes of nitroalkene reduction by PETN reductase under aqueous conditions. Inset: Time course of ab-
sorbance changes upon substrate reduction. The arrows indicate the direction of spectral change. (a) (Z)-2-(4’-chlorophen-
yl)-1-nitropropene [(Z)-4c]; (b) (E)-2-(4’-chlorophenyl)-1-nitropropene [(E)-4c]; (c) (Z)-1-(4’-chlorophenyl)-2-nitropropene
[(Z)-5b]; (d) (E)-1-(4’-chlorophenyl)-2-nitropropene [(E)-5b].
shown in Figure 1; spectral data for all other com- the (Z)-isomers of substrates 4. This demonstrates
pounds are in Supporting information Figure S3). The that PETNR substrate specificity extends to heteroar-
apparent preference of PETN reductase for substrates omatic derivatives with potential to vary the substitu-
under biotransformation conditions (reaction to com- ents at Cb.
plete substrate depletion) can be analysed by compar-
ing the 50% depletion time of each substrate (t50; both (Z)- and (E)-isomers of 1-(4’-chlorophenyl)-2-ni-
Supporting Information Table S1). tropropene 5b were very similar, with rates compara-
Unlike b,b-disubstituted nitroalkenes, the t50 of
PETN reductase showed a clear preference for the ble to the substrate (Z)-4a. The presence of a methyl
(Z)-b,b-disubstituted nitroalkenes 4a–d compared to group at Ca in substrate (E)-5a resulted in an approxi-
their respective (E)-isomers as seen by an approxi- mate 75-fold increase in apparent rate compared to
mate 24- to 45-fold increase in the t50values (Support- the equivalent substrate (E)-4a which has the methyl
ing Information Table S1). The preference for both group at Cb. The absence of a methyl group at Cb may
(Z)- and (E)-4a, c, d in terms of aromatic ring sub- have contributed to the increased reaction rate by al-
stituent decreased in the order: Br>Cl>H [~F for tering the orientation of substrate binding and/or di-
(E)-4b]. This effect was less pronounced with the (E)- minished steric hindrance. Detailed kinetic and struc-
substrates, as the apparent rates varied by only 2-fold. tural analyses are required to determine if these
When the phenyl ring of the substrate (E)-2 was re- trends are due to binding effects and/or the electron-
placed by thiophene [(E)-3], the turnover rate in- withdrawing effects of the substituted aromatic rings
creased by 5- to 15-fold compared with (E)-2, al- on the Cb.
though the rate with (E)-3 was 3- to 7-fold lower than
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Intermediates Formed during Nitroalkene Reduction
Stereochemical Outcome of the Reaction
The spectral studies performed to determine the rates Enoate reductases from the OYE family[6,30] produce
of reduction of nitroalkenes 1–5 provide some insight opposite enantiomers from (E)- and (Z)-enoates and
into the reaction carried out by PETN reductase. By enals, enabling a substrate-based control of the ste-
using a NADPH-regenerating system[27] we were able reochemical outcome of the reaction. In contrast, the
to follow the spectral changes during substrate reduc- reduction of (E) and (Z)-isomers a- or b-alkyl-b-aryl-
tion in the absence of NADP+/NADPH spectral inter- nitroalkenes catalysed by bakerꢁs yeast[14,16] or crude
ference. Previous studies with wild-type/Y196F extracts of Clostridium sporogenes,[15] proved to be an
OYE1[27] and morphinone reductase (MR)[28] showed enantioconvergent process. There is less information
that hydride transfer and protonation steps during re- about the stereochemical course of the reduction of
duction of 1 are decoupled, in contrast to the reaction (E)- and (Z)-nitroalkenes with purified enzymes of
with 2-cyclohexen-1-one in which these steps are con- the OYE family, although their enantioselectivity
certed.[29] The decoupling makes it possible to monitor seems to differ widely (Scheme 3). OPR3, YqjM and
the accumulation of the nitronate intermediate, which NCR reduce (E)-4a to the (S)-enantiomer of 9a, al-
results in an increase in absorbance in the region of though OPR3 and YqjM exhibit rather low enantiose-
238 nm. As expected, nitronate accumulation and lectivities.[8–10] In contrast, OYE1-3 and OPR1 pro-
slow decay was seen during the reduction of 1-nitro- duce the (R)-enantiomer of 9a with 83% ee and 98%
cyclohexene with PETN reductase (Supporting Infor- ee, respectively.[8,9] Therefore, different enantiomeric
mation Figure S3a) similar to wild-type OYE1.[27] Ap- products can be obtained by judicious selection of the
parent nitronate accumulation and decay was also biocatalyst.
seen with substrates (E)-5a and (E)-5b (Supporting
In order to investigate the stereochemical outcome
Information Figure S3i and Figure 1d, respectively), and the enantioselectivity of the reaction, the biore-
both of which contain a methyl group at the carbon ductions were performed in a biphasic reaction
to be protonated (a-carbon). Furthermore, substrate system (isooctane/phosphate buffer pH 7.0, 40:60, v/v;
decay of (E)-5b proceeded at a linear rather than ex- Table 2) in the presence of the glucose 6-phosphate
ponential rate. The decay at 238 nm with the other dehydrogenase NADP+/NADPH cofactor regenera-
substrates (Supporting Information Figure S3b–h; Fig- tion system.[15] This allowed us to conduct reactions at
ure 1a–c) closely followed the decrease in absorbance higher concentrations of the substrate and enabled
due to hydride transfer, suggesting that the hydride facile monitoring of the reaction by chiral HPLC and
transfer and protonation steps may be concerted with GC-MS.
these substrates.
Similarly to OPR3, YqjM and NCR, the reduction
Studies with Y186F mutant of OYE1[27] and MR[28] with (E)-isomers of 4a–d by PETN reductase led to
showed either a considerable delay or absence, re- the formation of the products (S)-9a–d (Table 2), al-
spectively, of decay of the nitronate intermediate of though with only moderate and variable enantiopurity
1-nitrocyclohexene attributed to protonation by water (63–89% ee). Furthermore, by-products were formed,
rather than by the enzyme. Similarly, previous studies which led to decreased product yield (18–58%,
with PETN reductase showed that water is the most
likely proton donor during the reduction of 2-cyclo-
hexene-1-one.[24] The similarity with the kinetics of ni-
tronate accumulation and decay suggest that 1-nitro-
cyclohexene (1) and nitroalkenes 5 are reduced via a
similar mechanism. However, studies with wild-type
OYE1[27] showed that nitronate accumulation with 1-
nitrocyclohexene does not rule out the possibility of
an enzymatic protonation step. Therefore, compara-
tive studies of reductions of nitroalkenes 1–5 with
Y186F PETN reductase mutant[24] are required to de-
termine whether Y186 plays a role in nitronate proto-
nation with these substrates, or if this apparent decou-
pling of the hydride transfer and protonation steps
with (E)-5a and (E)-5b is due to essentially non-cata-
lytic protonation by water.
Scheme 3. Asymmetric bioreduction of a,b-disubstituted ni-
troalkenes by OYE enzymes. OYE1-3=old yellow enzymes
1–3; NCR=NCR enoate reductase from Zymomonas mobi-
lis; OPR1 and 3=12-oxophytodienoate reductase isoforms
from Lycopersicon esculentum; YqjM=from Bacillus subti-
lis.[8–11]
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Table 2. Reduction of nitroolefins 1–5 by PETN reductase under biphasic reaction conditions.[a]
Number Substrate
lmax[b] [nm] Time[c] [days] Conversion[d,e] [%] Yield[d,e] [%] Product[f] ee[e] [%]
1
264
319
357
2
2
1
100
>99
>99[g]
82
6
7
8
N/A
N/A
N/A
(E)-2
(E)-3
100[g]
100
(E)-4a
(Z)-4a
(E)-4b
(E)-4c
306
208
312
310
3
3
3
7
100[h]
100[h]
100
58[h]
89[h]
86
(S)-9a
(S)-9a
(S)-9b
(S)-9c
89[h]
96[h]
63
70
18
72
(Z)-4c
(E)-4d
(Z)-4d
221
314
224
4
7
7
100
73
91
35
76
(S)-9c
(S)-9d
(S)-9d
96
75
83
>99
(E)-5a
(E)-5b
316
321
230
3
7
7
100[h]
93
93[h]
84
(R)-10a
(R)-10b
(R)-10b
14[h]
54
(Z)-5b
88
80
60
[a]
Conditions: The reactions contained potassium phosphate buffer (50 mM KH2PO4/K2HPO4 pH 7.0, 9.2 mL) nitroalkene
(1.7 mM in 4.8 mL anaerobic isooctane), NADP+ (20 mM), glucose 6-phosphate (14–20 mM), glucose 6-phosphate dehy-
drogenase (10 units), deoxygenated PETN reductase (2 mM) and sec-butylbenzene (25 mL). Reactions were agitated at
200 rpm for 1–7 days at 308C in an anaerobic cabinet. N/A=not applicable.
Maximal absorbance wavelength of the substrate used to monitor reduction.
Time of biphasic reaction in days.
[b]
[c]
[d]
Conversions and yields were determined by HPLC from calibration curve of each standard substrate and products and
corrected using sec-butylbenzene as internal standard.
[e]
[g]
[f]
Chiralcel OD-H.
Absolute configuration assigned by comparison with literature data.[15]
Phenomenex C-18. [h] Chiralcel OJ.
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Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond
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Table 2). These products were tentatively assigned by after 5 days the conversion was only about 70%, with
GC-MS to be their equivalent oxime, aldehyde (Nef only 18% yield of the product (S)-9c (72% ee)
reaction product) and alcohol. In contrast, the reduc- (Table 2). In the case of a,b-disubstituted nitroal-
tion of (Z)-isomers of 4 by PETN reductase gave (S)- kenes, reduction of both (Z)- and (E)-isomers of ni-
enantiomers of the corresponding nitropropanes 9 in troalkenes 5b proceeded with a similar apparent rate,
almost quantitative yields and with high enantiopurity enantioselectivity (54–60% ee) and yield (80–84%).
(ꢂ96% ee). Thus, the reduction of (E)- and (Z)-iso- This suggests that the substrates bind in a similar
mers of substrates 4 proceed in a stereoconvergent manner, possibly via one or more of the highly con-
manner.
served H181/H184 residues, as suggested for other
Reduction of the a,b-disubstituted nitroalkenes OYE enzymes (Scheme 4).[32] The detection of the op-
(E)-5a, b (Scheme 2, Table 2), using PETN reductase posite enantiomeric products in the reduction of (E)-
generated the products (R)-10a and b with a much 4a–d and (E/Z)-5a–b suggests more than one binding
lower optical purity (14% and 54% ee, respectively), mode of the substrates is possible (Scheme 4).
although the reaction rate and yield were generally
It has been consistently observed that whereas the
very good. The reduction of the (Z)-isomer of 5b was reduction of b,b-disubstituted nitroalkenes of struc-
stereoconvergent with the reduction of its (E)-coun- ture 4 usually proceeds in a highly enantioselective
terpart and it led to the formation of the product (R)- fashion, the a,b-disubstituted nitroalkenes 5 are re-
10b in a comparable yield, but with a slightly higher duced with poor enantioselectivity.[6] Low product
optical purity (60% ee). Furthermore, small amounts enantiopurity in the reduction of a,b-disubstituted ni-
of by-products were detected during reduction of the troalkenes 5 was found in the reactions with C. sporo-
compounds 5, which were identified by GC-MS as genes[15] and bakerꢁs yeast.[16] Initially, this had been
their respective arylpropanones (Nef reaction prod- attributed to racemisation of the product under the
ucts).
reaction conditions[6,14] but it is now known that the
The stereoconvergent course of the reduction of exchange of the Ca proton is relatively slow.[15,16] Pre-
both isomers of the substrates 4 and 5 is in striking vious studies showed that a 5-day incubation of the
contrast with the stereodivergence of the reduction of product in D2O yielded only about 5% proton ex-
a,b-unsaturated carboxylic acids and carbonyl com- change, which does not fully account for the observed
pounds, which depends on geometry of the substrate low enantiopurity of the products 10.[15] Since the
(E/Z-isomers).[6,11,31] This unusual behaviour with ni- enantiopurity of the product of the reduction of the
troalkenes could be the result of one or more of the substrates 5 depends on the protonation step of Ca,
following: (i) the substrate orientation in the active the low enantioselectivity of PETN reductase could
site of an enzyme is not determined by the interaction be also due to slow non-enantiospecific interconver-
of the nitro group with H181 and/or H184 as with sion of the aci-form of the corresponding nitronic acid
enoates; (ii) PETN reductase may only react with one of the bioreduction mechanism (Scheme 1b).[15,16,27] At
isomer of the substrate and non-catalytic E/Z-isomeri- present the protonation step is not fully understood,
sation of the substrates ensures the supply of the pre- however literature data suggest that protonation is
ferred isomer in the reaction and/or (iii) the reaction the rate-limiting step for bioreductions of nitroal-
proceeds via trans addition[9] of Hꢀ and H+ with the kenes.[27,33] Thus nitronate anions themselves can act
a- and b-carbons of both (E)- and (Z)-substrate bind- as ambident nucleophiles (Scheme 1b), which can be
ing in the same relative orientation (b- and a-carbon attacked from both soft and hard nucleophiles such as
above and below FMN N5, respectively).
carbon and oxygen, respectively. These can attack soft
To investigate this, we followed the time course of or hard electrophiles respectively and such behaviour
reduction of pairs of (E)- and (Z)-isomers of nitroal- can be seen in the Nef reaction, for example.[34]
kenes 4c (Figure 2a and b) and 5b (Figure 2c and d). Therefore, one can also expect the formation of both
It should be noted that there was a low rate of non- a nitroalkane (protonation of Ca) and a respective ni-
enzymatic E/Z-isomerisation of each substrate under tronic acid (protonation of O), which will slowly equi-
the reaction conditions with no PETN reductase pres- librate to the optically inactive nitroalkane
ent (Supporting information Figure S4). Substrate (Scheme 1b). As postulated by Swiderska and Stew-
(Z)-4c (Figure 2a) was converted to the product (S)- art, the pKa values of the nitronate intermediate and
9c (96% ee) within 4 days. The sample of (Z)-4c con- the amino acid residue can make proton transfer ther-
tained 7% of the (E)-isomer due to non-catalytic Z- modynamically unfavourable.[35]
to E-isomerisation prior to the reaction. The concen-
In this context, the formation of the Nef reaction
tration of the (E)-isomer decreased, despite the con- by-products during the reduction of nitroalkenes
tinuous isomerisation of (Z)-4c, indicating that either could be a result of an ambident character of nitro-
the (E)-isomer was being consumed or that (E)- to nate intermediates;[27] however it cannot be concluded
(Z)-isomerisation was occurring. In contrast, while whether or not this process involves enzymatic cataly-
the conversion of (E)-4c initially proceeded quickly, sis. It is noteworthy that similar to the OYE family,
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Figure 2. Time course of the PETN reductase-catalysed reduction of (Z)- and (E)-isomers of 2-(4’-chlorophenyl)-1-nitropro-
~
*
pene 4c (a and b, respectively) under biphasic conditions. Substrates: & =(Z)-4c; =(E)-4c; product: =(R)-9c. Time
course of the PETNR-catalysed reduction of (Z)- and (E)-isomers of 1-(4’-chlorophenyl)-2-nitropropene 5b (c and d, respec-
~
*
tively) under biphasic conditions. Substrates: & =(Z)-5b; =(E)-5b; product: =(R)-10b. The % ees throughout the reac-
tions were fairly consistent with that of the final product (% ee was 52–60% and 53–56% for (Z)-5a and (E)-5a, respectively)
and dropped by only 1% per day.
Scheme 4. Theoretical model for the selectivity of PETN reductase with (E)-4 (a) and (E)-5 (b), adapted from[45]. AA=
amino acid residue/s modelled to interact with the substrate.
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Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond
FULL PAPERS
flavin nitroalkene oxidases[36] catalyse the transforma- pound 1-nitrocyclohexene (1) binds, as seen in the
tion of nitroalkenes to the respective aldehydes and PETN reductase Y186F structure (pdb code
ketones, in what is an enzymatic equivalent of a Nef 2ABB).[24] Inspection of the electron density for the
reaction.[34] Other enzymes capable of catalysing such cyclohexene ring revealed that all atoms except for
reactions are horseradish peroxidase,[37] propionate-3- C3 lie approximately in one plane. This suggests that
nitronate oxidase from Penicillium atrovenetum,[38] the substrate is bound in two different orientations
and 2-nitropropane dioxygenase from Neurospora (in a 1:1 ratio) corresponding to the two different
crassa.[39] These latter reactions also involve the for- half-chair conformations of the 1-nitrocyclohexene
mation of nitronates, which may suggest that Nef by- ring. The two binding modes are related by approxi-
product formation by PETNR may be a catalytic mately 1808 rotation of the cyclohexene ring about
rather than a chemical decomposition of the inter- the N7-C1 bond with an inversion in the half-chair
mediate; however, this hypothesis needs to be con- conformation (Figure 3a). Such multiplicity of binding
firmed by appropriate mechanistic studies. Interest- modes has been shown to occur in PETN reductase
ingly, the enzyme CYP83B1 from Arabidopsis thali- with various non-physiological substrates and inhibi-
ana catalyses oxime conversion to thiohydroximic tors[23] due to the relatively large size of the active site
acid via the formation of a nitronate intermediate in and the presence of few direct interactions between
the biosynthetic pathway of glucosinolates,[40] which is the substrates/inhibitors and the enzyme.
analogous to the reverse of the reaction catalysed by
1-Nitrocyclohexene (1) binds to the si-face of the
PETN reductase. While the exact mechanisms of side- flavin and is stabilised by a hydrogen bond to H184,
product formation in PETN reductase are unknown, with van der Waals contacts with H181, T26, W102,
such comparisons with other known enzymes catalys- Y186, and Y351. The electronegative nitro group is
ing similar reactions may give insight into possible stabilised by proximity to positively charged H181
mechanisms of action. Additionally, PETNR is known and H184. These residues are known to play a similar
to catalyse additional types of reactions seen by its role with other substrates such as steroids, 2-cyclohex-
ability to degrade nitroaromatic explosives such as enone, picric acid and TNT.[18,22–24] In one binding
TNT and picric acids.[18,22] Thus, whilst PETN reduc- mode, the b-carbon atom C2 (activated for nucleo-
tase is a promiscuous biocatalyst, there is a need for philic attack through resonance stabilisation with his-
(i) detailed evaluation of the mechanism and (ii) cau- tidine H181 and/or H184)[18] is only 3.7 ꢂ away from
tion in inferring mechanistic similarities between vari- the FMN nitrogen atom N5. Superposition of this
ous OYE family members and between bioreduction structure with the PETN reductase-picric acid com-
of various a,b-unsaturated compounds and their iso- plex shows that C2 of the cyclohexene moiety is
mers.
bound in a very similar position to that of the b-
carbon of picric acid[18] (results not shown). Further-
ꢀ
more, the angle between atoms N10
G
A
ꢀ
Structure of the PETN Reductase Complex with 1-
Nitrocyclohexene 1
(FMN) C2(1-nitrocyclohexene) is 1058, which is close
to optimal for hydride transfer.[41] This suggests that
the reaction mechanism for the reduction of the C=C
We determined the structure of the 1-nitrocyclohex- double bond of 1-nitrocyclohexene proceeds via a
ene-bound PETN reductase to 1.34 ꢂ resolution to direct nucleophilic attack by hydride from the flavin
assist the modelling of aryl-ntiropropenes 4 and 5. We N5 atom at the Cb (C2) position of the substrate. In
used 1-nitrocyclohexene because the low solubility contrast, the second binding mode for 1-nitrocyclo-
and apparent high Kd values for the latter substrates hexene (Figure 3a) is thought to be non-productive as
prevent structure determination of complexes using the double bond of the cyclohexene ring is not in a
X-ray crystallography. The data collection and refine- suitable orientation and distance from the flavin N5.
ment statistics are found in Supporting Information
Analysis of the productive binding mode of 1-nitro-
Table S2. The structure was isomorphous with the cyclohexene showed that the Ca (C1) of the substrate
previously determined apo-enzyme and substrate- is only 3.2 ꢂ away from the hydroxy group of Y186
ꢀ
ꢀ
bound complexes with 2-cyclohexenone and ste- with an angle CZACHTNUGTRENNUG(Y186) OHACHTUNGTNER(NUGN Y186) C1(1-nitrocyclo-
roids,[18,22–24] except for a minor change in the back- hexene) of 1258. This suggests a possible role of Y186
bone conformation of a surface loop near the sub- as a potential proton donor to Ca (C1) in the C=C
strate entry point (residues T273 to P280).
double bond reduction, as seen in OYE,[42] but further
The electron density maps are consistent with 1-ni- kinetic studies are required to determine if water or
trocyclohexene being bound close to the flavin ring Y186 is the proton donor.
(modelled with a partial occupancy of ~60%) in the
enzyme active site (Figure 3a). There was also evi-
dence for partial occupation of a chloride ion and iso-
propyl alcohol molecule at the position where com-
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Figure 3. Active site of PETN reductase containing
bound substrate. The figures were generated in
Pymol.[56] All residues are shown as atom-coloured
sticks with green and yellow carbons for protein
and FMN, respectively. Hydrogen bonds are indi-
cated by dotted lines (grey lines indicate the bond
is behind the side chain). (a) Stereo view of the
2jFo jꢀjFc j electron density map contoured at 1 s
and final atomic model of the active site of 1-
bound PETN reductase. The catalytic and non-pro-
ductive positions for 1 are shown as atom-coloured
sticks with pink carbons and grey lines, respective-
ly. Dual models of the (b) (E)- and (c) (Z)-isomers
of 4c and 5b bound in the active site of PETN re-
ductase. The substrates (E)- and (Z)-4c are shown
as atom-coloured sticks with magenta and blue car-
bons for models 1 and 2, respectively. The sub-
strates (E)- and (Z)-5b are shown as atom-col-
oured lines with magenta and blue carbons for
models 1 and 2, respectively. The hydrogen bonds
are colour-coded to indicate the substrate and
model, with large and small dots for (E/Z)-4c and
(E/Z)-5b substrates, respectively.
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FULL PAPERS
Structure-Based Models of Nitroalkene Enzyme-
Substrate Complexes
This is likely to have a significant effect on the turn-
over rate of the enzyme, and suggests that catalysis
(with non-physiological substrates) may require a
We generated two models each of nitropropenes 4 metastable enzyme-substrate complex rather than
and 5 bound to the active site of PETN reductase, preferential binding of the more stable complex/es
where we maintained a close to ideal distance and ori- observed crystallographically.[24] This could explain
entation of the b-carbon of the substrate to N5ACHTNUTRGNEUNG(FMN) why modelling the most likely catalytically active con-
(3.71 ꢂ and 1058, respectively), with minimal clashes formations based on previously published structures
with surrounding residues. Models 1 and 2 were based does not necessarily coincide with maximal substrate-
on the positions of the 1-nitrocyclohexene and pub- enzyme interactions for substrates 4 and 5. Addition-
lished picric acid-bound structures, respectively, in the ally, knowledge is lacking as to the relative contribu-
productive binding conformation in PETN reductase tions of the interactions between the b-carbon and
(Figure 3b and c).[23,24,43] Model 1 of the p-Cl substitut- FMN N5 compared to interactions with His181/184,
ed nitroalkene substrates (E)-4c and (E)-5b (Fig- and the role these interactions play in determining
ure 3b) showed the nitro group of the substrate to be the optimal binding mode/s of the substrates for effi-
in a similar position as the nitro group of the active 1- cient catalysis.
nitrocyclohexene-PETN reductase complex, where it
Although we do not have a crystal structure for the
can potentially form hydrogen bonds with residues complexes of PETN reductase with b,b- or a,b-disub-
H181 and H184.[23] These hydrogen bonds are also stituted nitroalkenes 4 and 5, the enantioselectivity of
seen in the PETN reductase structures containing the reaction can be predicted from existing knowl-
picric acid and 2,4-dinitrophenol (between histidine edge such as (i) the currently accepted OYE flavin-
and OH groups).[18] In these models, the aromatic mediated anti-hydrogenation mechanism, (ii) allowan-
rings of the substrates and Y68 are within van der ces made for water-mediated protonation, (iii) knowl-
Waals contact, although no p-p stacking is observed. edge of the dual binding modes of substrates in
There appears to be a loss of the interaction between OYE1[44] and PETN reductase[23] and (iv) substrate
the OH groups of Y68 and Y186, with further poten- modelling in the active site. A theoretical model for
tial van der Waals contacts with T26, Y186 and S132. the enantioselectivity of PETN reductase and OYE1
The side chain of Y351 was reoriented to accommo- with p-hydroxybenzaldehyde was proposed recent-
date the aromatic ring of the substrate.
ly,[45] and has been adapted here for a/b-alkyl-b-aryl-
In model 2 of substrates (E)-4c and (E)-5b (Fig- nitroalkenes (Scheme 4). The mechanism of C=C
ure 3b) a potential loss of interaction between the double bond reduction by OYE1, based on the bind-
nitro group O2 and H184 NE2 was seen as well as a ing of p-hydroxybenzaldehyde, suggests that a,b-unsa-
putative interaction modelled between the nitro turated substrates stack above the reduced flavin with
group O1 and T26 OG. Both models for substrate Cb aligned optimally with the N5 for hydride transfer
(E)-4c suggest that the methyl group (Cb) is posi- from below.[42,46] Tyr196 (Y186 in PETN reductase)
tioned in close proximity to the side chain of T26, lies above the substrate and donates a proton to the
which may hinder productive substrate binding. This Ca in OYE1. Assuming a model where protons are
may explain why the nitroalkenes (E)-4a–d are added to the side of the substrate facing away from
poorer substrates compared with (E)-2, since the the flavin, whether from Y186 or water, substrates
latter substrate differs by the lack of the methyl (E)-4a–d bound in models 1 and 2 (Scheme 4a and
group at the Cb.
Figure 3b) are likely to form the (S)-enantiomeric
Model 1 of the substrates (Z)-4c and (Z)-5b was product (chiral Cb). Substrates (Z)-4a, b bound in a
similar to those for substrates (E)-4c except for minor similar orientation are also likely to produce the same
shifts in position and a reorientation of the aromatic enantiomer (S) of the product (Scheme 4b and Fig-
ring (Figure 3c). This enabled a favourable FMN N5- ure 3c). This is in agreement with our data that show
substrate carbon distance and orientation to be main- that both (E)- and (Z)-isomers of 2-aryl-1-nitropro-
tained, but resulted in the loss of potential interac- penes are reduced to form products (S)-9.
tion/s of the nitro group with H181 and H184 (Fig-
Previous docking studies with nitroalkene (E)-4a in
ure 3c). However, model 2 of these substrates main- OPR1 and OPR3 predicted two substrate binding
tained the nitro group O2 and H184 NE2 interaction, modes resulting in the (R)- and (S)-enantiomeric
with an additional potential interaction between the products, respectively,[8] with the likelihood of occu-
Cl atom of the substrate and S132 OG atom (Z)-5b.
pancy dependent on the architecture of the active site
The structures of the progesterone-[23,24] and 1-nitro- of each OYE family member. PETN reductase con-
cyclohexene-PETN reductase complexes have shown formations for binding the substrates (E)- and (Z)-4c
that the enzyme can bind substrates and inhibitors in that would generate the (R)-enantiomeric product are
non-productive conformations, with potentially a low less likely to be populated significantly due to clashes
occupancy of the catalytically active conformations. between the aromatic ring of the substrates and the
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protein (results not shown). Nevertheless, the nitroal- compounds likely to fit in the active site. Modelling
kenes (E)-4a–d, unlike their (Z)-counterparts, were techniques based on high-resolution substrate-bound
reduced with lower enantioselectivity (63–89% ee). structures in combination with knowledge of the reac-
This may be accounted for by a small occupancy of tion mechanism and idealisation of critical FMN-sub-
the (E)-substrate bound in an alternative binding strate distances/orientations enables the rational
mode, resulting in a different stereochemical out- design of substrates by the introduction of new func-
come. E- to Z- or Z- to E-isomerisation studies under tional groups as well as in some cases predicting the
aqueous conditions (up to 3 h) showed the presence enantioselectivity of the reaction. It could also poten-
of oxidised PETN reductase (NADPH absent) did tially generate new improved biocatalysts by predict-
not significantly affect the isomerisation rates (results ing specific mutations of PETN reductase necessary
not shown), suggesting the reduced enantioselectivity for more specific biocatalysis of industrially useful
with substrates (E)-4a–d and (E/Z)-5a–b is unlikely to synthons. This work emphasises the need for (i) de-
be due to isomerisation of the enzyme-bound sub- tailed evaluation of the mechanism of catalysis and
strate. Therefore, the near 100% ee with (Z)-sub- (ii) caution in inferring mechanistic similarities in
strates suggests either a tighter control of the hydride structurally related enzymes.
transfer and/or constraints within the active site that
restrict the range of catalytic binding modes. The cur-
rent mechanism of catalysis of OYEs suggests a trans Experimental Section
addition of Hꢀ and H+,[9] with the protonation step
catalysed by Y186.[8] However, if the protonation step General Remarks
is water-mediated for some substrates, it may be pos-
NMR spectra were recorded on 300 MHz or 400 MHz spec-
sible that reduction of such substrates with PETN re-
trometers and referenced to the solvent, unless stated other-
ductase could proceed via syn addition, which would
wise. The chemical shifts are reported in ppm and the cou-
generate both enantiomeric products with substrates
(E/Z)-5a, b.
pling constants (J) are given in hertz (Hz). GC-MS analysis
was performed using a DBWAX column (30 m, 0.25 mm,
0.25 mm thick, JW Scientific) and an MS unit equipped with
an ion trap. IR spectra were recorded neat on NaCl plates.
HPLC analysis was performed using an instrument equipped
with a UV detector. Yield and conversion, as well as ee of
the products 6 and 7, were determined by HPLC using Chir-
alcel OD-H or Chiralcel OJ columns (ø 4.6 mmꢃ250 mm)
and for the compound (E)-2 using Phenomenex Gemini C-
Conclusions
We have shown that PETN reductase has a broad
substrate specificity for nitroalkene reduction, and ex-
hibits excellent enantioselectivity for reduction of
(Z)-isomers of b,b-disubstituted nitroalkenes 4. This
makes the enzyme potentially useful in the produc-
tion of industrially useful enantiopure nitroalkanes.
This provides a further example of catalytic promiscu-
ity of the enzyme, which was initially detected by its
ability to reduce nitroaromatic explosives via the for-
mation of the Meisenheimer-hydride complex.[19,22]
This catalytic flexibility is likely due, at least in part,
to the relatively large active site, which allows the
enzyme to bind a range of substrates with considera-
ble size variation and to orientate them appropriately
for reduction of different functional groups. Most im-
portantly, PETN reductase is a highly expressing,
cheap to produce, robust enzyme, making it especially
useful as a prospective industrial biocatalyst. Lastly, it
crystallises readily leading to atomic resolution struc-
tures which can be used to analyse reaction selectivity
and mechanism. This information can be used to pre-
dict mutations that may enhance catalytic properties,
rather than having to rely solely on random mutagen-
esis to generate useful biocatalysts.
18 (5 mm,
ø 4.6 mmꢃ250 mm, MeOH:H2O, 85:15, v/v,
1 mLminꢀ1). Absolute configuration of the nitroalkanes was
determined by comparison of chiral chromatography data
with the literature.[15] Preparative HPLC, used for the sepa-
ration of (E) and (Z) isomers, was carried out on an HPLC
system equipped with a UV detector, using a Rainin Dyna-
max 60 A silica 41.4 mmꢃ25 cm column coupled with a
41.4 mmꢃ5 cm guard column. UV-visible data were record-
ed with either a spectrophotometer situated in a glove box
operating at less than 1 ppm oxygen or under anaerobic con-
ditions with a diode array spectrophotometer. For isomerisa-
tion of nitroalkenes (E)-4 and (E)-5, a medium-pressure
photochemical ultra-violet lamp was used. The sonication
reactions were carried out in an ultrasound bath at highfield
frequency 35 kHz. All reactions were monitored by GC-MS
and HPLC. Substrates 1–3, product 6 and other reagents
were obtained from commercial sources, and the solvents
were of analytical grade.
Synthesis of Substrates and Reference Materials
Compounds (E)-4a–d, (Z)-4c, (E)-5a and b and model race-
mic nitroalkanes 7–10 were synthesised according to proce-
dures previously described.[15] (Z)-Isomers of nitroalkenes
4a, 4d and 5b were synthesised by isomerisation of the re-
spective (E) isomers by UV light, as previously described.[15]
(Z)-1-Nitro-2-phenylpropene [(Z)-4a]: Yellow oil; puri-
The use of a structure-driven modelling approach
in predicting potential substrates for biocatalysis is an
invaluable tool as it theoretically reduces the amount
of substrate screening required, and instead targets fied by chromatography (hexane/Et2O, 95:5); 1H NMR
2800
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Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond
FULL PAPERS
(CDCl3, 300 MHz): d=2.20 (d, J=1.5 Hz, 3H), 7.08 (q, J=
1.5 Hz, 1H), 7.19–7.23 (m, 2H), 7.40–7.42 (m, 3H);
13C NMR (CDCl3, 75 MHz): d=24.1, 126.3, 128.5, 128.8,
134.4, 137.2, 148.4; MS (EI): m/z=163 (1%, M+), 115
(100%); HR-MS (CI): m/z=197.0243, calcd for [M]+
C9H8NO2Cl: 197.0238; IR (neat): n=1340, 1519, 1636 cmꢀ1.
(Z)-2-(4’-Bromophenyl)-1-nitropropene [(Z)-4d]: Yellow
oil; purified by chromatography (hexane/Et2O, 95:5);
1H NMR (CDCl3, 300 MHz): d=2.18 (d, J=1.4 Hz, 3H),
7.07 (s, 1H), 7.08 (d, J=8.2 Hz, 2H), 7.53 (d, J=8.2 Hz,
2H); 13C NMR (CDCl3, 75 MHz): d=23.9, 123.1, 128.0,
131.8, 134.8, 136.0, 147.1; MS (EI): m/z=243 (5%, M+), 241
(5%, M+), 115 (100%); HR-MS [ESI(ꢀ)]: m/z=239.9671,
calcd for [MꢀH]+ C9H7NO2Br: 239.9666; IR (neat): n=826,
1010, 1338, 1521, 1637 cmꢀ1.
KH2PO4/K2HPO4 pH 7.0). Absorption change of the di-
thionite-reduced enzyme (15–20 mM) was monitored contin-
uously at the flavin absorption maxima (464 nm) with a
range of substrate concentrations at 258C. The concentra-
tions of substrates were always at least 10-fold greater than
the enzyme concentration, ensuring pseudo-first order con-
ditions. Each data point is the average of at least 5 transi-
ents. The transients were fitted to a double exponential plot
(single exponential plot for 4) from which observed rates
were determined using the rapid equilibrium formalism of
Strickland et al[47] [Eq. (1)] for the following kinetic scheme
[Eq. (2)].
(Z)-1-(4’-Chloro-phenyl)-2-nitro-propene [(Z)-5b]: Pale
yellow crystals: 1H NMR (CDCl3, 300 MHz): d=2.36 (s,
3H), 6.44 (s, 1H), 7.17–7.20 (m, 2H), 7.31–7.34 (m, 2H);
13C NMR (CDCl3, 75 MHz): d=23.9, 123.1, 128.0, 131.8,
134.8, 136.0, 147.1; HR-MS (EI): m/z=197.0241, calcd for
[M]+ C9H7NO2Cl: 197.0238; anal. calcd for C9H8ClNO2: C
54.70, H 4.08, Cl 17.94, N 7.09; found: C 54.58, H 4.05, N
7.07; IR (neat): n=867, 1356, 1490, 1525 cmꢀ1.
Steady State Kinetics
Spectrophotometric Studies on Bioreduction of
Purified PETN reductase was prepared as described previ- Nitroalkenes 1–5
ously.[23] All kinetics measurements were performed within
Biotransformations in homogeneous, aqueous reaction mix-
an anaerobic glove box (Belle Technology Ltd) under a ni-
trogen atmosphere (<5 ppm oxygen). The following extinc-
tion coefficients were used to calculate the concentration of
substrates and enzyme: NADPH (e340 =6,220Mꢀ1 cmꢀ1); 1-
nitrocyclohexene (1; e270 =6,100Mꢀ1 cmꢀ1); (E)-2-nitro-1-
phenylethene [(E)-2; e320 =15,500Mꢀ1 cmꢀ1]; (E)-2-thienyl-1-
nitroethene [(E)-3; e364 =17,900Mꢀ1 cmꢀ1]; and PETN reduc-
tase (e464 =11.3ꢃ103 Mꢀ1 cmꢀ1). PETN reductase is stable in
air, but was deoxygenated by passage through a BioRad
10DG column equilibrated in anaerobic reaction buffer to
avoid unproductive flavin reoxidation during nitroalkene re-
duction.
Steady state measurements were performed using the
enzyme monitored turnover method[26] which was originally
developed for the reactions catalysed by glucose oxidase.
Data were collected in an Applied Photophysics SX.18MV
stopped-flow spectrophotometer. Reactions were performed
in potassium phosphate buffer (50 mM KH2PO4/K2HPO4
pH 7.0) containing NADPH (200 mM) and substrate (0–
500 mM). Enzyme reduction (10 mM) was monitored contin-
uously at 464 nm until completely reoxidised at multiple
substrate concentrations. The experiments were analysed
using kinetic constants calculated as previously described.[26]
tures were performed using a Jasco UV/VIS spectrophotom-
eter. The reductions were carried out according to a modifi-
cation of the protocol of Meah and Massey,.[27] under anae-
robic conditions in potassium phosphate buffer (50 mM
KH2PO4/K2HPO4 pH 7.0) containing NADP+ (20 mM), glu-
cose 6-phosphate (20 mM), glucose 6-phosphate dehydro-
genase (10 units), substrate (50–100 mM) and PETN reduc-
tase (50–200 nM) at 258C. The presence of the enzyme glu-
cose 6-phosphate dehydrogenase was necessary to recycle
NADP+ to the electron donor NADPH required for PETN
reductase flavin reduction. The reaction was followed by pe-
riodically scanning the reaction mixture between 200 and
700 nm.
Bioreduction of Nitroalkenes 1–5 at Analytical Scale
in Biphasic Systems
Reactions were carried out anaerobically in 30-mL screw-
top vials sealed with PTFE-silicon septa. Each reaction mix-
ture (12 mL) contained potassium phosphate buffer (50 mM
KH2PO4/K2HPO4 pH 7.0) containing nitroalkene (1.7 mM)
dissolved in 4.8 mL anaerobic isooctane, PETN reductase (~
3 mM), NADP (20 mM) glucose 6-phosphate (14–20 mM),
glucose 6-phosphate dehydrogenase (10 units) and sec-butyl-
benzene (25 mL), the latter as the internal standard for chro-
matography. Reactions were agitated at 200 rpm for 1–7
days at 308C in an anaerobic cabinet. The organic layer was
analysed by HPLC, and also by GC-MS to verify the identi-
ty of the product(s). Enantiomeric excess, yields and conver-
sions were determined by HPLC using Chiralcel OD-H or
Chiralcel OJ columns (ø 4.6 mmꢃ250 mm, hexane:i-PrOH,
90:10, v/v, 1 mLminꢀ1). Products formed from compound
(E)-2 were analysed using a Phenomenex Gemini C-18
Rapid Reaction Kinetics of the Oxidative Half
Reaction
Oxidative half reaction kinetic experiments were performed
anaerobically using an Applied Photophysics SX.18MV
stopped-flow spectrophotometer. The transients were ana-
lysed using non-linear least squares regression analysis on
an Acorn Risc PC microcomputer using Spectra kinetics
software (Applied Photophysics). PETN reductase was ti-
trated with sodium dithionite to the two-electron level then
mixed with substrate in potassium phosphate buffer (50 mM
Adv. Synth. Catal. 2008, 350, 2789 – 2803
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
2801

Helen S. Toogood et al.
FULL PAPERS
column (5 mm, ø 4.6 mmꢃ250 mm, MeOH:H2O, 85:15, v/v,
Merz–Sing–Kollmann (MK) scheme using the keyword/
value pair ꢄpop=mkꢁ, this comprises fitting of atomic charg-
es to reproduce the molecular electrostatic potential (ESP)
around the van der Waals envelope. As the latter methods
do not recognise C-halide bonds, the minimised structures
were further refined using the structure idealisation function
of REFMAC5[50] to normalise the bond angles and distances
of the substrates and to remove minor clashes.
1 mLminꢀ1).
Crystallogenesis and Data Collection
Crystals of oxidised PETN reductase were grown using the
sitting-drop method as described previously.[23] The crystal
complex of oxidised PETN reductase with substrate 1 was
produced by soaking these crystals in mother liquor contain-
ing 30 mM of 1-nitrocyclohexene 1. The soaked crystals
were flash-frozen in liquid nitrogen in the absence of addi-
tional cryoprotectant, and a full 1.3 ꢂ X-ray diffraction data
set was collected from a single crystal at the European Syn-
chrotron Radiation Facility (Grenoble, France) on Station
ID 14.1 (wavelength 0.93 ꢂ; 100 K) using an ADSC CCD
detector.
Acknowledgements
We thank Ben Sattelle for his help in preparing the initial
energy minimisation models and Chandrani Mukherjee for
assistance in some of the analytical and synthesis work. This
work was funded by the UK Biotechnology and Biological
Sciences Research Council via grants BBE0107171 and
BBD0028261. AR is a Wellcome Trust Career Development
Fellow, DL is a Royal Society University Research Fellow
and NSS is a BBSRC Professorial Research Fellow.
Structure Determination and Refinement
Data were processed and scaled using the programs
MOSFLM[48] and Scala.[49] The structure was solved via mo-
lecular replacement using the coordinates for the PETN re-
ductase/picric acid complex[18] (PDB entry 1VYR) with
picric acid and water molecules removed. Positional and ani-
sotropic B-factor refinement was performed using
REFMAC5,[50] with alternate rounds of manual rebuilding
of the model in TURBO-FRODO.[51] The positions of water
molecules were found automatically using REFMAC com-
bined with ARP/wARP.[52] The final model was refined to
1.34 ꢂ resolution giving a final Rfactor of 0.114 and Rfree of
0.145. The atomic coordinates and structure factors (pdb
code 3F03) have been deposited in the Protein Data Bank,
Research Collaboratory for Structural Bioinformatics, Rutg-
ers University, New Brunswick, NJ (http://www.rcsb.org/).
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2803