Biocatalytic asymmetric alkene reduction: Crystal structure and characterization of a double bond reductase from Nicotiana tabacum

Article abstract of DOI:10.1021/cs300709m

The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B₄ dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP⁺ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.

Full text of DOI:10.1021/cs300709m

Research Article
pubs.acs.org/acscatalysis
Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and
Characterization of a Double Bond Reductase from Nicotiana
tabacum
David J. Mansell,†,⊥,§ Helen S. Toogood,†,‡,§ John Waller,†,‡ John M. X. Hughes,†,‡ Colin W. Levy,†,‡
John M. Gardiner,†,⊥ and Nigel S. Scrutton*,†,‡
†Manchester Institute of Biotechnology, ⊥School of Chemistry, and ‡Faculty of Life Sciences, University of Manchester, Manchester,
U.K.
S
* Supporting Information
ABSTRACT: The application of biocatalysis for the asymmetric
reduction of activated CC is a powerful tool for the
manufacture of high-value chemical commodities. The bio-
catalytic potential of “-ene” reductases from the Old Yellow
Enzyme (OYE) family of oxidoreductases is well-known;
however, the specificity of these enzymes toward mainly small
molecule substrates has highlighted the need to discover “-ene”
reductases from different enzymatic classes to broaden industrial
applicability. Here, we describe the characterization of a flavin-
free double bond reductase from Nicotiana tabacum (NtDBR),
which belongs to the leukotriene B4 dehydrogenase (LTD)
subfamily of the zinc-independent, medium chain dehydrogen-
ase/reductase superfamily of enzymes. Using steady-state kinetics
and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-
unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like
substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic CC
double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family
member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE “-ene” reductase activity and,
coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray
crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-
bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the
reactivity, stereochemistry, and specificity of this enzyme.
KEYWORDS: double bond reductase, asymmetric alkene reduction, biocatalysis, crystal structure, Nicotiana tabacum
considerable potential for exploitation in industrial biocatalysis;
however, the substrate specificity of these enzymes is generally
confined to small molecules, which limits the scope of their
applicability. This highlights the importance of targeting “-ene”
reductases from different enzymatic classes, which could
potentially reduce alkene substrates that are not acted upon
by OYEs because there is likely to be a significantly altered
substrate binding site.
A potential source for “-ene” reductases may exist in the zinc-
independent, medium chain dehydrogenase/reductase (MDR)
superfamily, or more specifically, the leukotriene B4 dehydro-
genase (LTD, MDR002) subfamily.5 This subclass of enzymes
do not contain any bound coenzyme and have been shown to
catalyze the reduction of the CC double bonds of α,β-
INTRODUCTION
■
The intelligent use of enzymes for the production of high-value
fine chemicals, pharmaceuticals, and agrochemical intermedi-
ates is at the forefront of the drive toward the use of mild and
ecologically sustainable manufacturing techniques.1 This is
largely due to the unmatched chemo-, regio-, and stereo-
specificity that can be achieved when using enzyme-catalyzed
reactions with either whole cell preparations or purified
enzymes.1c,2 The biocatalytic asymmetric reduction of activated
CC bonds has received particular attention because of the
potential for production of chiral compounds with up to two
new stereogenic centers.3 Recent work has demonstrated the
biocatalytic potential of the Old Yellow Enzyme (OYE; E.C.
1.6.99.1) family of oxidoreductases in the reduction of activated
α,β-unsaturated alkenes, including α,β-unsaturated aldehydes,
ketones, nitroalkenes, and carboxylic acids.3,4 These FMN-
containing, NAD(P)H-dependent oxidoreductases are an
exceptionally versatile group of biocatalysts and offer
Received: November 2, 2012
Revised: January 18, 2013
Published: January 21, 2013
© 2013 American Chemical Society
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Research Article
Scheme 1. Reactions Catalyzed by the Reductases (a) PtPPDBR and At5g16970, (b) AOR or At5g16970, and (c) NtDBR.6−9
unsaturated aldehydes, ketones, and carboxylic acids.6 For
example, phenylpropenal double bond reductase (PtPPDBR)
from Pinus taeda catalyzes the NADPH-dependent reduction of
the α,β-unsaturated bond of phenylpropenal aldehydes
(Scheme 1a).7 A similar alkenal double bond reductase
(At5g16970) was discovered in Arabidopsis thaliana, which
reduces the C7−8 alkene group of p-coumaryl- and coniferyl
aldehydes (Scheme 1a) and 4-hydroxy-(2E)-nonenal (4-HNE)
(Scheme 1b).6 In rat liver, the detoxification of 4-HNE is
catalyzed by the NAD(P)H-dependent alkenal/one oxidor-
eductase (AOR) to form 4-hydroxynonanal (4-HNA; Scheme
1b).8 In humans, 4-HNE is a product of lipid peroxidation and
can lead to apoptosis, affect cell signaling pathways, and form 4-
HNE-protein adducts found in Alzheimer plaques.6
A BLAST search of LTD subfamily members identified a
putative double bond reductase from Nicotiana tabacum
(NtDBR). This enzyme has a high amino acid sequence
identity to other LTD members: At5g16970 (71%),6 2-alkenal
reductase (Dbr1) from Artemisia annua (67%),10 (+)-pulegone
reductase (PulR) from Mentha piperita (66%),11 and PPDBR
(41%).7 This enzyme was originally misclassified as an NAD+-
dependent allyl-alcohol dehydrogenase (NtADH) because the
enzyme isolated from tobacco leaves showed alcohol
dehydrogenase (ADH) activity toward carveol.12 However,
this activity was absent in the recombinant form of the enzyme
and purported to be due to the absence of suitable post-
translational modification capabilities of the host Escherichia
coli.9 NtDBR was later described as a pulegone reductase
because of its ability to hydrogenate the exocyclic CC bond
of (R)- and (S)-pulegone to produce menthone and
isomenthone isomers (Scheme 1c).13 In light of the moderate
yields achieved for the latter conversion, we have chosen to
refer to the enzyme as a generalized double bond reductase
(NtDBR). Because menthone and isomenthone have important
industrial applications in the production of synthetic
peppermint oils and bases, we decided to further investigate
the biocatalytic potential of recombinant NtDBR. This paper
describes our kinetic characterization of the substrate and
cofactor specificity of NtDBR and determination of its regio-
and stereospecificity toward typical LTD alkenal/one substrates
and commercially useful compounds. We also determined the
crystal structure of NtDBR both in isolation (holo) and in
enables us to expand our understanding of the catalytic
mechanism and substrate binding modes and drive future
structure-driven site-saturated mutagenesis studies to further
expand the biocatalytic potential of this enzyme.
EXPERIMENTAL SECTION
■
Gene Synthesis and Cloning. The protein sequence for
the allyl alcohol dehydrogenase (accession number: Q9SLN8)
from N. tabacum was obtained from UniProt (http://www.
uniprot.org). The gene sequence was designed and synthesized
by GenScript (USA), incorporating codon optimization
techniques of rare codon removal for optimal expression in E.
coli. The gene was cloned into pET21b (Novagen) via NdeI/
XhoI restriction sites, without a stop codon, to incorporate a C-
terminal His6-tag. The construct was transformed into the E.
coli strain BL21(DE3)pLysS (Stratagene) for soluble protein
overexpression according to the manufacturer’s protocol.
Enzyme Production and Purification. The NtDBR
construct was grown in Terrific broth medium (ForMedium)
containing glycerol (0.4%), ampicillin (100 μg mL−1), and
chloramphenicol (34 μg mL−1). Cultures were incubated at 37
°C until OD600 nm reached 0.5, followed by a 16 h induction
with isopropyl-β-D-1-thiogalactopyranoside (IPTG; 10 μM) at
30 °C. Cells were harvested by centrifugation at 5000g for 10
min at 4 °C. Cell pellets were resuspended in lysis buffer (50
mM Tris, pH 8.0) containing the EDTA-free complete protease
inhibitor cocktail (Roche), DNase I (0.1 mg mL−1), and
lysozyme (1 mg mL−1) and stirred for 20 min at 4 °C. Cells
were disrupted by sonication (Sonics Vibra Cell), followed by
extract clarification by centrifugation for 60 min at 26 600g.
The supernatant was filtered (0.2 μm) and applied to a Ni2+
Sepharose high-performance column (40 mL; GE Healthcare)
preequilibrated in buffer A (50 mM Tris, pH 8.0, containing
300 mM NaCl, 10 mM imidazole, and 10% glycerol). The
column was washed with five column volumes of buffer A,
followed by a step elution of NtDBR into buffer B (50 mM
Tris, pH 8.0, containing 300 mM NaCl, 300 mM imidazole,
and 10% glycerol). The enzyme was dialyzed against buffer C
(50 mM Tris, pH 8.0, containing 10% glycerol) and reapplied
to the Ni2+ Sepharose column, preequilibrated as before. The
column was washed as above, and the enzyme was eluted in a
linear gradient (300 mL) to buffer D (50 mM Tris, pH 8.0,
containing 300 mM NaCl, 500 mM imidazole, and 10%
glycerol). The enzyme was dialyzed against buffer C as above,
complexes with NADP(H) (binary)
alkene substrate 4-
hydroxy-3-methoxycinnamaldehyde (HMCA) (ternary). This
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a
Table 1. Steady-State Kinetic Parameters of NtDBR with a Variety of Substrates
a
Standard reactions were performed in 50 mM K2HPO4/KH2PO4 pH 7.3 + 1 mM alkene, 5% ethanol and 100 μM NADPH. Data in parentheses are
the rates (s−1) with 1 mM alkene rather than kcat. ND = not determined. Alkenes not reduced by NtDBR under steady-state conditions were methyl
α-methyl-trans-cinnamate, methyl trans-cinnamate, trans-cinnamonitrile, trans-cinnamic acid, trans-4-phenyl-3-buten-2-one, 2-cyclohexenone, 3-
methyl-2-cyclohexen-1-one, 1-cyclohexene-1-carboxylic acid, 1-cyclohexene-1-carbonitrile, 2-cyclohexen-1-ol, 2-cyclopenten-1-one, 2-methyl-2-
cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, 3-methyl-2-butenal, citral, (5R)- and (5S)-carvone, ketoisophorone, isophorone, 2-
methylmaleimide, N-phenyl-2-methylmaleimide, itaconic acid, mesaconic acid, citraconic acid, crotonitrile, (S)-perillyl alcohol, and β-bromostyrene.
1
2
Reactions were performed in the presence of 500 μM nitrocyclohexene, which is below the expected Km for this substrate. Estimated kinetic
parameters are based on fits of partial Michaelis−Menten plots due to substrate concentration range limitations.
concentrated by ultrafiltration, and flash frozen in liquid
nitrogen for storage at −80 °C. Purity was assessed by SDS−
PAGE, and the concentration was determined using the
Bradford and extinction coefficient methods (ε205 = 30.86 mg
mL−1).14
Research Collaboratory for Structural Bioinformatics, Rutgers
University, New Brunswick, NJ (http://www.rcsb.org/). The
interactions involved in the dimeric interfaces were analyzed by
the protein interfaces, surfaces, and assemblies service PISA at
the European Bioinformatics Institute (http://www.ebi.ac.uk/
msdsrv/prot_int/pistart.html).18
Crystallogenesis and Data Collection. Crystals of the C-
terminally His6-tagged NtDBR were obtained by sitting drop
vapor diffusion at 20 °C after a period of 7 days of incubation.
Drops were prepared by mixing equal volumes (2 μL) of
mother liquor (100 mM KH2PO4/citrate buffer, pH 4.0−5.0,
containing PEG 300 (35−50%)) and NtDBR (25 mg mL−1 in
10 mM Tris, pH 7.0). Crystals of the holoenzyme were flash-
frozen in liquid nitrogen in the absence of any additional
cryoprotectant. Additional crystals were soaked in mother
liquor containing (A) NADPH or (B) NADP+ and 4-hydroxy-
3-methoxycinnamaldehyde to form the binary and ternary
complexes, respectively. All ligand concentrations were at half
saturation, and crystals were flash frozen in liquid nitrogen in
the absence of any additional cryoprotectant. Full holo-NtDBR
(1.94 Å), binary-NtDBR (1.9 Å), and ternary-NtDBR (2.1 Å)
X-ray diffraction data sets were collected from single crystals at
the Diamond Light Source (Oxford, UK).
Structure Determination and Refinement. Data were
processed and scaled using the programs MOSFLM15 and
Scala.16 The structures were solved via molecular replacement
using the program MolRep16 and the coordinates for a Swiss-
MODEL-generated pdb file of NtDBR (http://swissmodel.
expasy.org). This model was generated on the basis of the
known three-dimensional structure of Arabidopsis alkenal
double bond reductase At5g16970 (PDB 2J3H).6 Positional,
TLS (two domains per monomer), and isotropic B-factor
refinement was performed using REFMAC5,17 with alternate
rounds of manual rebuilding of the model and water addition in
COOT.16 Data and refinement statistics are shown in Table 2.
The atomic coordinates and structure factors (codes: 4HFJ,
4HFM and 4HFN, for the holo, binary, and ternary structures,
respectively) have been deposited in the Protein Data Bank,
Steady-State Kinetics. Steady-state kinetic analysis was
performed on a Cary UV-50 Bio UV/vis scanning spectropho-
tometer using a quartz cuvette (1 mL; Hellma) with a 1 cm
path length. Reactions (1 mL) were performed in buffer (50
mM Tris, pH 7.4) containing NADPH (0.1 mM), alkene (1
mM added as concentrated stocks in ethanol to a final
concentration of 5%) and enzyme (10 nM−4 μM). Reactions
were followed continuously by monitoring NADPH oxidation
at 340 nm (ε340 = 6220 M−1 cm−1) for 1 min at 25 °C.
Reactions with alkenes that absorb at 340 nm were monitored
at 365 nm (ε365 = 3256 M−1 cm−1). Standard units of activity (1
μmol NADPH min−1 mg−1 NtDBR after protein purification)
were determined in buffer (50 mM KH2PO4/K2HPO4, pH 5.4)
containing 100 μM NADPH and 500 μM 1-nitrocyclohexene at
25 °C. Typical batches contained 2.01−2.47 U/mg enzyme. All
comparative reactions were performed using the same enzyme
batch. To determine the optimal reaction conditions and
steady-state reaction parameters (kcat, Km, and Ki), the reaction
pH (5.0−7.4), buffer (KH2PO4/K2HPO4 or KH2PO4/
K2HPO4/citrate), enzyme (10 nM−4 μM), and substrate
concentrations (10 μM−10 mM) were varied. In cases that
reaction constraints prevented a full steady-state analysis, the
specificity factor (kcat/Km) was determined from the initial
Michaelis−Menten slope. Substrate concentrations were mostly
determined using their respective extinction coefficients
previously described,19 or as follows: trans-2-nonenal (ε227
=
15 600 M−1 cm−1), 1-octen-3-one (ε214 = 8500 M−1 cm−1),
methyl crotonate (ε210 = 12 500 M−1 cm−1), (R)-pulegone (ε263
= 6310 M−1cm−1).
Biotransformation with NtDBR. All reagents were of
analytical grade. The alkenes were dissolved as stock solutions
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a
Table 2. X-ray Crystallographic Data Collection and Refinement Statistics
b
parameters
holo NtDBR
binary NtDBR
ternary NtDBR
space group
C2
C2
C2
cell dimensions
a, b, c (Å)
88.57, 148.74, 67.73
90.0, 115.59, 90.0
42.13−2.00 (1.99−2.00)
5.5 (66.4)
88.20, 149.84, 66.61
90.0, 115.53, 90.0
60.1−1.9 (2.00−1.90)
8.8 (36.8)
88.86, 149.86, 66.21
90.0, 115.96, 90.0
37.47−2.01 (2.06−2.01)
7.2 (45.9)
α, β, γ (o)
resolution (Å)
c
Rmerge (%)
I/σI
15.5 (2.3)
11.7 (3.6)
9.4 (2.3)
completeness (%)
redundancy
98.8 (98.0)
97.8 (95.2)
98.1 (99.3)
4.8 (4.8)
5.8 (4.3)
3.2 (3.2)
no. reflections
unique reflections
275 755
346 704
161 936
57 956
59 835
51 397
R
work/Rfree
RMS deviations
bond angles (o)
bond lengths (Å)
Ramachandran plot
18.2/22.7 (25.0/28.6)
17.3/20.6 (21.8/25.8)
17.2/22.1 (22.2/27.3)
1.541
0.018
1.612
0.016
1.769
0.020
allowed region (%)
97.0
2.7
98.3
1.7
97.3
2.7
additionally allowed region (%)
a
b
PDB codes for Holo-NtDBR, binary-NtDBR and ternary-NtDBR are 4HFJ, 4HFM, and 4HFN, respectively. Highest resolution shell is shown in
c
parentheses. Rmerge = Σhkl Σi |Ii(hkl) − [I(hkl)]|/Σhkl Σi Ii(hkl), where Ii(hkl) is the intensity of the ith observation of unique reflection hkl.
Redundancy = total number of reflections/total unique reflections. Rwork = Σ||Fobs| − |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are observed and model
structure factors, respectively. Rfree was calculated by using a randomly selected set (5%) of reflections.
in ethanol, then diluted to 5 mM in the reactions (2% final
solvent concentration). (i) Standard biotransformations: Stand-
ard reactions (1.0 mL) were performed aerobically in
phosphate buffer (50 mM KH2PO4/K2HPO4 pH 6.4)
containing alkene (5 mM), NtDBR (2.0 μM), NADP+ (10
μM), glucose (15 mM), and glucose dehydrogenase (GDH; 10
U). The reactions were shaken at 30 °C for 4 or 24 h at 130
rpm, and terminated by extraction with ethyl acetate (0.9 mL)
containing an internal standard. The extracts were dried using
anhydrous MgSO4 and analyzed by GC to determine the
percent yield, percent conversion, and enantiomeric excess.
Products were identified by comparison with authentic
standards. (ii) Effect of pH: Reactions were performed as
described for the standard biotransformations, except a variety
of buffer compositions and final pH values were used (50 mM
KH2PO4/K2HPO4, pH 6.0−7.4; or 50 mM KH2PO4/K2HPO4/
citrate, pH 5.4−6.0). (iii) Reduction of (5R)-pulegone in the
presence of auxiliaries: Reactions (1.0 mL) were performed as
described under the standard biotransformations conditions
with the addition of Triton X-100 (0.01% or 0.1%) BSA (15
μM) or in the presence of BSA (15 μM) alone. (iv)
Biotransformations with PETN reductase: PETN reductase
was produced and purified as described previously.20 Reactions
(1.0 mL) were performed anaerobically in buffer (50 mM
KH2PO4/K2HPO4 pH 7.0) containing PETN reductase (2.0
μM). Reactions were set up within an anaerobic glovebox
(Belle Technology Ltd.) under a nitrogen atmosphere (<5 ppm
oxygen) using anaerobic solvents. Prior to addition, PETN
reductase was deoxygenated by passage through a BioRad
10DG column equilibrated in anaerobic reaction buffer. All
other details were the same as those described under the
standard biotransformation conditions.
optimum was found to be 5.4 with 1-nitrocyclohexene (1a)
(Supporting Information Figure S1), in contrast to prior
reported reaction conditions of pH 7−8.9,12,13 Given that
typical biotransformation reactions proceed around neutral pH,
we determined the steady-state kinetic parameters of NtDBR at
both pH 5.4 and 7.3 (Table 1). In many cases, full steady-state
kinetic parameters could not be determined because of either
limited alkene solubility (trans-2-nonenal, 4a), significant
alkene absorbance at 340/365 nm at higher substrate
concentrations [1a and trans-cinnamaldehyde (6a)], or the
detection of near-zero-order kinetics with respect to substrate
concentration down to 10−20 μM [1-octen-3-one (5a) and
NADPH at pH 7.3]. In these cases, either the specificity factor
(kcat/Km) or the rate with 1 mM alkene was determined (Table
1).
NtDBR showed a higher preference for NADPH as the
hydride donor13 compared with NADH (>1600-fold). This is
not surprising, given the significant number of specific
interactions observed between the enzyme and the adenosyl
phosphate group in the binary and tertiary structures (vide
infra: Structural Analysis of NtDBR) (Figure 2B). This
preference for NADPH was also seen with Dbr110 and rat
AOR21 and may be a common feature of LTD enzymes.
Although full steady-state parameters could not be determined
for some substrates, NtDBR shows a high preference for 1-
nitrocyclohexene (1a) over typical LTD alkenal/one substrates
(Table 1). However, significant activity was detected with
alkenals: 2-methylpentenal (2a) and trans-cinnamaldehyde (6a)
at pH 5.4 (kcat/Km = 8.9 and 2.8, respectively), although the Km
is predicted to be higher (millimolar range) than with 1a. In
contrast, the Km values with AOR-like substrates trans-2-
nonenal (4a) and 1-octen-3-one (5a) are predicted to be in the
low micromolar range, similar to NADPH binding (results not
shown). They showed reaction rates (0.87 0.07 and 0.52
0.03 s−1, respectively) similar to 2a and 6a (0.36 0.02 and
0.80 0.03 s−1, respectively) at pH 7.3. In addition, substrate
RESULTS AND DISCUSSION
■
Substrate Specificity of NtDBR. The activity of NtDBR
toward a variety of activated alkenes was determined under
steady-state conditions (Table 1). Surprisingly, the pH
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inhibition was seen with 2a (apparent Ki = 2.6 0.6 mM) and
5a, although only at pH 5.4.
NtDBR is predicted to display tight binding with 4a and 5a
(zero-order kinetics down to 10 μM substrate; results not
shown); therefore, it is likely to be specific for long chain fatty
alkenals/ones. LTD family members commonly reduce the
substrate 4a, displaying tight binding kinetics, high rates, or
both. For example, At5g16970 showed both a high catalytic rate
(kcat = 51 s−1) and apparent tight binding (Km = 5.9 μM)
toward 4a.22 In contrast, enzymes Dbr1 and AOR showed
apparent tight binding (Km = 6.4/77.6 μM), but slower reaction
rates (kcat = <1/0.2 s−1) with 4a.10,21
A comparison of the kinetic parameters of NtDBR at pH 5.4
vs 7.3 shows an interesting trend of a decrease in both the
catalytic rate and Km with increasing pH (Table 1). This trend
could possibly be due to a change in the position of the
substrates α,β-unsaturated bond relative to the NADPH pro-4R
hydrogen at neutral pH, resulting in a tighter-binding, yet less
favored enzyme−substrate complex. Alternatively, the change
in the overall rate may be due to a decrease in the product(s)
KD, dissociation rates, or both. Further, fast reaction kinetics
studies are required to determine the mechanism of the pH
effects on the overall reaction kinetics.
Minor activity (<0.1 s−1) was seen under standard steady-
state conditions with alkene substrates: α-methyl trans-
cinnamaldehyde, 1-cyclohexene-1-carboxaldehyde, methyl crot-
onate, (R)-pulegone, and dimethyl itaconate. No reduction was
seen with other activated alkenes, such as maleimides, citral,
(5R)- or (5S)-carvone, (S)-perillyl alcohol, and substituted
cyclohexenones and cyclopentenones (see Table 1 footnote for
full list). The poor reactivity with (R)-pulegone was
unexpected, given the detection of significant activity in prior
studies by others (kcat = 3.3 s−1, Km = 1.4 mM);9 however, the
earlier studies were carried out under biotransformation
conditions (pH 7.0, 3 mM NADPH, 1 mM dithiothreitol,
0.2% Triton X-100), and the presence of Triton-X-100 is
known to dramatically enhance the catalytic rate of NtDBR.13
In addition, previous comparative studies of alkene reduction
by pentaerythritol tetranitrate reductase and thermostable Old
Yellow Enzyme (TOYE) showed that significant alkane product
could be detected in biotransformations with substrates poorly
reduced under steady-state conditions.19a,23 Therefore, longer
biotransformation reactions (24 h; 5 mM alkene) allow a more
comprehensive investigation of the substrate range of alkene
reduction.
Structure Analysis of NtDBR. We determined the X-ray
crystal structures of the holo-, binary (NADP(H)-bound), and
ternary (NADP+ and 4-hydroxy-3-methoxycinnamaldehyde-
bound) NtDBR complexes. The data collection and refinement
statistics can be found in Table 2. The asymmetric unit of each
structure contains two protein molecules, related by a
noncrystallographic 2-fold axis (C2 symmetry). The overall
structure of NtDBR exhibits a typical MDR-like protein fold
(Figure 1A), arranged as a functional homodimer (Figure
1B).24 Each monomer is composed of two domains (Figure
1A): a substrate binding or catalytic domain (residues 1−135
and 304−343) and a nucleotide-binding domain (residues
136−303). The catalytic domain contains three α-helices and
eight β-strands, with the majority of the β-strands forming a
twisted partial β-barrel-like structure, as seen in At5g16970
structures (Figure 1C).6 In contrast, the nucleotide-binding
domain contains a typical alternating pattern of α-helices (αA-
αG) and β-strands (βA-βF), forming a Rossman fold seen in
Figure 1. The overall structure of NADP(H)-bound NtDBR (a)
monomer and (b) dimer. Each structure is shown as a cartoon with a
gradient from blue at the N-terminus to red at the C-terminus. The
secondary structure elements are labeled in part a, including indicating
where β6 is present in other structures. NADP(H) is shown as atom
colored sticks with blue carbons. (c) ClustalW alignment of the
protein sequences of NtDBR and At5g16970 double bond reductases.
The α-helices and β-sheets are indicated as red and blue bars/labels,
respectively. The dotted box highlights the residues making up the
nucleotide-binding domain. Residues highlighted in bold text are
involved in NADP(H)-binding. Residues highlighted with yellow
shading are within van der Waals distance to 4-hydroxy-3-
methoxycinnamaldehyde in the ternary complex.
many nucleotide-binding enzyme structures. The active site is
formed in a groove located at the interface of the two domains,
with a minor contribution of residues from the neighboring
monomer.
Using the program DALILITE,25 each of the three structures
was shown to be virtually identical (rmsd of 0.5−1.0 Å) with Z
scores of 49.6−54.5 (331 residues aligned by Cα). NtDBR is
also very similar to the structure of another LTD subfamily
member At5g16970 (rmsd of 0.8−1.1 Å; Z score = 47.4−
50.8).6 Further details of the subtle differences among the three
NtDBR structures and At5g16970 can be found in the
Supporting Information.
Dimeric Interface. The dimeric interface is composed of
residues located in the nucleotide-binding region (αE-βF) of
each subunit (Figure 1C; Supporting Information Table 2).
These secondary structure elements are oriented antiparallel
with respect to each monomer chain, particularly in the case of
strand βF (Figure 1A and 2B). This gives the appearance of an
extended β-sheet across the dimer interface, similar to the
At5g16970 structure.6 An analysis of the dimeric interface of
each structure gave complex formation significance scores
(CSS) of 0.5−1.0, with interface areas of 1019−1058 Å2. This is
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Figure 2. (A) Structure of the dimeric interface of NADP(H)-bound NtDBR. (B) NADP(H)-binding mode of the binary NtDBR crystal structure.
The omit |Fo| − |Fc| map of NADP(H) in subunit A is shown as a blue mesh. (C) Crystal structure of the active site of the NADP+- and HMCA-
bound NtDBR crystal structure. The omit |Fo| − |Fc| map of HMCA is shown as a blue mesh. (D) Superimposition of the alkene/alkane binding
pockets in the ternary structures of NtDBR and At5g16970 (pdb: 2J3J and 2J3F). Residues surrounding the HMCA binding site are shown as atom
colored sticks with green and blue carbons for subunits A and B, respectively. NADP+ and HMCA/4-HNE are shown as atom-colored sticks with
yellow and teal/orange carbons, respectively. Residue numbers are color-coded according to the enzyme residue carbon color. In each part, the
protein is shown as green and teal cartoons or sticks, for subunits A and B, respectively. NADP(H) is shown as atom colored sticks with yellow
carbons. Water molecules are shown as red spheres. Interactions are shown as dotted lines (orange lines refer to interactions occurring behind the
atoms shown).
indicative of a functional interaction,18 consistent with the
observation that wild-type and recombinant NtDBR exist as a
homodimer in vitro.9,12
surrounding the NADP(H) binding site are slightly altered in
position compared with the binary and ternary structures. The
side chain positions of residues Y206, N332, and S136 are
significantly different between the holoenzyme and the
NADP(H)-bound structures. In the case of S136, this is to
prevent a clash of this residue with the nicotinamide moiety of
NADP(H).
The position and orientation of NADP(H) in subunit A of
the binary and ternary structures are virtually identical. In
contrast, the mode of NADP(H) binding in subunit B of the
binary complex differs significantly because of a reorienting of
atoms O5B and C5B of the adenosine moiety (Figure 2B). The
position of the nicotinamide moiety in these structures and the
At5g16970 binary and ternary complexes is highly conserved;6
however, the orientation of the remaining cofactor atoms can
vary significantly between subunits and between enzymes. In
the case of the At5g16970 ternary complex, the cofactor
reorients beginning at the D-ribose moiety relative to NtDBR,
although the positions of the terminal adenine base and
phosphate group are similar in both structures.6 Further details
about the more subtle differences in the NADP(H) binding can
be found in the Supporting Information.
The interactions within the dimeric interface are composed
of mainly hydrogen bonds between backbone (e.g., G281 N−
I277 O) or side chain atoms (e.g., H267 ND1−263 OG1;
Supporting Information Table 1). In some cases, pairs of the
same residues of each subunit interact via backbone N and O
atoms (V266 and M279). Salt bridges also play a role in the
interface, with multiple interactions seen between residues
R276 and D286 of each subunit (Supporting Information Table
1). Residues E280 (OE2) and R276 (O) of subunits A and B,
respectively, also interact via the presence of a bridging water
molecule. Interestingly, the βF region (residues 277−286) in
LTD enzymes is known to be involved in NADP(H) binding
(Figure 2A),6 suggesting the dimeric state may be important for
either substrate binding, enzyme activity, or both.
NADP(H)-Binding Site. Both the binary and ternary crystal
structures showed clear |Fo| − |Fc| electron density for the
NADP(H) (Figure 2B). The cofactor binds in the cleft joining
the catalytic and nucleotide-binding domains in an extended
conformation (Figure 1), similar to other LTD enzymes. In the
holoenzyme, this cleft is filled with water molecules, and the
backbone positions of surface regions α1 and β7-α3
Ternary Complex. The crystal structure of NtDBR in the
presence of the alkene substrate derivative 4-hydroxy-3-
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methoxycinnamaldehyde (HMCA) and product NADP+
showed clear |Fo| − |Fc| electron density for the presence of
NADP+ and a second ligand in subunit A only (Figure 2C, D);
however, the electron density for the putative propenal ring
substituent of HMCA was partially disordered and nonplanar
with respect to the aromatic ring. The close proximity of the
side chain of L283 to the propenal group of HMCA may be
causing it to be oriented in a less favored nonplanar
conformation. Alternatively, the second ligand could potentially
be the alkane product derivative 4-hydroxy-3-methoxyhydro-
cinnamaldehyde (HMHCA), with substrate hydrogenation
occurring via X-ray exposure during data collection. Therefore,
we are unable to ascertain the exact nature of the second ligand
and have modeled in HMCA because this was the compound
soaked into the crystals.
(Table 1); therefore, we studied the overall effect of pH on the
NtDBR-catalyzed reduction under biotransformation condi-
tions. In addition, changes in pH have also been shown to
influence the enantiopurity of products that are subject to
racemization under the reaction conditions.19a The assays were
performed aerobically against two representative substrates, 1-
nitrocyclohexene (1a) and methyl-trans-2-methylcinnamalde-
hyde (3a) (5 mM substrate; 2 μM NtDBR), for 4 and 24 h at
pH 5.4, 6.4, and 7.4 using a NADP+/glucose dehydrogenase
(GDH) cofactor recycling system as the source of reducing
equivalents (Table 3).
Table 3. Influence of pH on the Reduction of 1a and 3a by
a
NtDBR
Unfortunately, we cannot directly infer the likely active
binding mode of HMCA because the substrate/product is
bound in an orientation with the propanal group at the protein
surface, distal from the NADP+ nicotinamide moiety (Figure
2C). Therefore, the ternary complex is likely an inhibitory
complex, although the position of HMHCA aids us in
determining which residues are likely to be involved in forming
the alkene substrate binding pocket. In contrast to NADP(H)
binding, there are no hydrogen bonds between HMHCA and
the protein. This is consistent with the observation that there is
little change in the position of residues lining the alkene-
binding pocket between the structures, with the exception of
mobile surface residues F285 and E29. Instead, there are direct
polar interactions between the 4-OH group (O11) of HMHCA
and both the N1N and O2D atoms of NADP+ (Figure 2C;
Supporting Information Table 3). These interactions are likely
responsible for the orientation of HMHCA in an inhibitory
complex. The binding pocket contains six hydrophobic residues
within van der Waals distance of HMHCA, including two
conserved NADP(H)-binding residues (Y55 and Y258), and
two nonconserved residues from subunit B (F270 and I273).
A superimposition of the ternary structures of NtDBR and
At5g16970 (NADP+ and p-coumaryl aldehyde (PCA) or 4-
HNE) showed significant differences in the second ligand
binding positions (Figure 2D). This is due to the binding of
PCA and 4-HNE in a likely active conformation to At5g16970.
A comparison of the residues lining the alkene-binding site in
both structures shows a high conservation in sequence identity
and residue position in most cases. Notable exceptions include
two phenylalanine-to-serine substitutions (NtDBR to
At5g16970; 285/287 and 270/272), which effectively introduce
additional polar residues into the latter enzyme. In the case of
F285, this residue clashes with PCA and would likely have to
reorient to allow the binding of cinnamaldehyde-like substrates
in an active conformation. Therefore, the presence of two
additional phenylalanine residues may restrict the size of the
binding pocket and impact on the enzyme substrate specificity.
Further site-directed mutagenesis work is required to confirm
the mechanistic role(s) of individual residues in substrate
binding and catalysis and confirm whether the dimeric form is
required for catalysis.
a
Conditions: reactions (1 mL) were performed aerobically in buffer
(50 mM KH2PO4/K2HPO4, pH 7.4 and 6.4; or 50 mM KH2PO4/
K2HPO4/citrate, pH 5.4) containing alkene (5 mM), NtDBR (2 μM),
NADP+ (10 μM), glucose (15 mM) and glucose dehydrogenase
(GDH; 10 U). The reactions were agitated at 30 °C for 4 or 24 h at
130 rpm. Conversion (conv) and yields were determined by GC
analysis on a DB-WAX column. The alkenes were added from stock
solutions in ethanol (2% final solvent concentration in the reactions).
The product obtained during the reduction of 3a was near racemic in
all cases.
The reduction of 1-nitrocyclohexene (1a) proceeded
efficiently at both pH 5.4 and 6.4, with complete conversion
of the substrate observed after 4 h; however, at pH 7.4, the
reduction was significantly slower and had failed to reach
completion after 24 h, consistent with the rates observed under
steady-state conditions (see above). Furthermore, poor product
yields were observed at pH 7.4, despite apparent high substrate
consumption, indicating decomposition of substrate, product,
or both. In contrast, the reduction of methyl-trans-cinnamalde-
hyde (3a) was most efficient at pH 6.4 and 7.4, with almost
identical results obtained at both pH values. Changes in pH did
not influence the stereochemical outcome, as near-racemic
product was obtained at all pH values. Therefore, the optimum
pH for reduction was considered to be pH 6.4, and all further
assays were performed at this pH.
NtDBR proved to be highly efficient in the reduction of
several typical LTD-like substrates: 2-methylpentenal (2a), 1-
octen-3-one (5a), and trans-cinnamaldehyde (6a), all of which
were completely reduced after 4 h (Table 4). In contrast, 1-
cyclohexene-1-carboxaldehyde (7a) and citral (8a) were
reduced with moderate and low yields, respectively. In terms
of stereoselectivity, the reduction of citral 8a [the citral used
here was a mixture of geranial (E)-8a and neral (Z)-8a in
∼65:35 ratio] is complicated by isomerization of this substrate
between its E/Z forms under the reaction conditions.19a Some
reduction of 4-hydroxy-3-methoxycinnamaldehyde (9a) was
observed, despite the apparent inhibitory binding conformation
observed in the tertiary crystal structure (Figure 2C). This
suggests that more than one binding conformations may exist
for this ligand.
Biotransformations with NtDBR. To fully evaluate the
biocatalytic potential of NtDBR and gain an insight into the
stereochemical outcome of these reactions, we studied the
reduction of a variety of compounds with different activating
groups and substitution patterns under biotransformation
conditions. Under steady-state conditions, a decrease in both
the catalytic rate and Km was observed with increasing pH
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a
Table 4. Reduction of Various Activated Alkenes by NtDBR Using an NADP+/GDH Cofactor Recycling System
a
Conditions: reactions (1 mL) were performed aerobically in buffer (50 mM KH2PO4/K2HPO4, pH 6.4) containing alkene (5 mM), NtDBR (2
μM), NADP+ (10 μM), glucose (15 mM), and glucose dehydrogenase (GDH; 10 U). The reactions were agitated at 30 °C for 4 or 24 h at 130 rpm.
The alkenes were added from stock solutions in ethanol (2% final solvent concentration in the reactions). Conversion (conv) and yields were
determined by GC analysis using a DB-WAX column. By chiral GC. Total yield of diastereoisomers [(2S, 5R)-menthone and (2R, 5R)-
isomenthone] By GC analysis using a Rtx-5 ms column. NtDBR did not catalyze the reduction of the following substrates: methyl trans-cinnamate,
b
c
d
e
dimethyl itaconate, methyl crotonate, (S)-perillyl alcohol, (5R)- and (5S)-carvone, and 2-methylmaleimide.
The reduction of the E and Z isomers of 2-phenyl-1-
nitroprop-1-ene (10a) proceeded with an opposite stereo-
chemical outcome: the reduction of (E)-10a furnished the R
product in optically pure form, whereas the reduction of (Z)-
10a generated the S product with poor enantioselectivity
(Table 4). This is in contrast to our previous observations with
the Old Yellow Enzyme, PETN reductase, which furnished the
same S enantiomer from the reduction of both (E)- and (Z)-
10a with moderate and high optical purity, respectively.19a In
the case of PETN reductase, this was attributed to multiple
substrate binding conformations for (E)-10a, with the
preference for the S product under anaerobic conditions.19a,20
Therefore, it is likely that in NtDBR, the E and Z isomers bind
predominantly in the same conformation, leading to an
enantiodivergent outcome. The lower ee values obtained
from the reduction of (Z)-10a may originate from isomer-
ization between the E/Z forms or a second, less favored,
binding conformation of the Z isomer in the NtDBR active site.
Significantly, we are now able to generate both enantiomers of
10b in optically pure form by using either PETN reductase or
NtDBR, eloquently highlighting the potential of this enzyme as
a complementary biocatalytic tool to classical OYE “-ene”
reductases. The related 1-phenyl-2-nitropropane (11b) was also
obtained under the reaction conditions, albeit in racemic form.
The low enantiopurity of 11b and other products possessing a
stereogenic center at the Cα position (2−3b) has been
attributed to the product racemization in aqueous media.19a,26
In such circumstances, a biphasic reduction system may be
employed to deliver improved product enantiopurity.27
Although some differences were seen in the enantioselectivity
of the reactions catalyzed by NtDBR vs PETN reductase, the
product yields from both reactions were similar with substrates
1a (both >99%) and 2a (96 vs 85%). More significant
differences in product yields were seen with substrates 8a (12%
vs 56%) and (E)-10a (93 vs 70%) for DBR and PETN
reductase, respectively. The reduction of (R)-pulegone (12a)
afforded (2S,5R)-menthone and (2R,5R)-isomenthone
(Scheme 1c) in moderate yield and in a ratio of ∼1:2. In
contrast, a putative second enzyme present in N. tabacum has
been shown to catalyze the reduction of (R)-12a with high
yields and stereoselectivity, producing almost entirely (2R,5R)-
isomenthone.28 Prior studies suggested an improvement in
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a
Table 5. Reduction of Various Activated (R)- and (S)-Perillaldehyde (13a) by NtDBR and PETNR
a
Conditions: NtDBR reactions (1 mL) were performed aerobically in buffer (50 mM KH2PO4/K2HPO4, pH 6.4) containing alkene (5 mM),
NtDBR (2 μM), NADP+ (10 μM), glucose (15 mM), and glucose dehydrogenase (GDH; 10 U). PETN reductase reactions (1 mL) were performed
anaerobically in buffer (50 mM KH2PO4/K2HPO4, pH 7.0) containing alkene (5 mM), PETNR (2 μM), NADP+ (10 μM), glucose (15 mM), and
glucose dehydrogenase (GDH; 10 U). All reactions were agitated at 30 °C for 4 or 24 h at 130 rpm. The alkenes were added from stock solutions in
b
ethanol (2% final solvent concentration in the reactions). Conversion (conv), yields, and cis/trans percentage were determined by GC analysis on a
DB-WAX column.
stereospecificity could be achieved for the reduction of (R)-12a
with recombinant NtDBR by the addition of bovine serum
albumin (BSA). In addition, the presence of Triton X-100, γ-
cyclodextrin, and casein were shown to improve the overall
conversion.13 Accordingly, we repeated the NtDBR-catalyzed
reduction of (R)-12a in the presence of Triton X-100 BSA
and in the presence of BSA alone. However, under our
conditions, no improvement in conversion or stereospecificity
was observed (results not shown). This may be due to
differences in the sequence or folding of the two protein
preparations. The originally published data13 were obtained
from protein expressed as a GST fusion, with the majority of
the tag removed by proteolysis. In contrast, our data were
obtained from C-terminally His6-tagged protein. It is possible
that the presence of different types of protein tags during
protein expression has affected the conformation of the protein,
leading to the observed differences in rate, stereospecificity, or
both.
Interestingly, NtDBR-catalyzed reduction of (R)- and (S)-
perillaldehyde (13a) generated the same major product from
both enantiomers, cis-dihydroperillaldehyde (13b) (Table 5).
This is not consistent with the net trans-hydrogenation
mechanism observed for OYE-catalyzed alkene reduction,
which is known to proceed via hydride transfer (from reduced
flavin, FMNH2) to the Cβ atom of the substrate and proton
transfer (from a conserved tyrosine residue or water) to the Cα
position of the substrate.3 Indeed, control experiments with
PETN reductase reflected net trans-hydrogenation, regardless
of the substrate’s absolute configuration, yielding trans-
dihydroperillaldehyde and cis-dihydroperillaldehyde from (S)-
and (R)-13a, respectively (Table 5). This unusual behavior has
previously been observed for another member of the LTD
subfamily, leukotriene B4 12-hydroxydehydrogenase (Ltb4dh),
and was subsequently shown to originate from a divergent
stereochemical pathway with (R)-perillaldehyde undergoing net
trans-addition of H2, whereas the reduction of (S)-peril-
laldehyde proceeded with net syn-addition of H2.29 This was
reported as the first observation of its kind by a single enzyme.
However, these results suggest it is highly likely that NtDBR
and potentially other members of the LTD family share this
unusual feature.
CONCLUSIONS
■
We have extended our study of “-ene” reductases to include
NtDBR, a member of the LTD subfamily of the MDR enzymes.
Substrate profiling, via steady-state kinetics and product
identification techniques, has demonstrated its regio- and
stereospecificity against a variety of α,β-unsaturated activated
alkenes. In addition to high activity toward typical LTD
substrates, NtDBR also catalyzed the reduction of several
classical OYE-like substrates. Significantly, NtDBR also
demonstrated activity toward non-OYE substrates (i.e.,
reducing the exocyclic CC double bond of (R)-pulegone)
as well as displaying the opposite enantioselectivity during the
reduction of substrates such as (E)-2-phenyl-1-nitroprop-1-ene
(10a) and (S)-perillaldehyde (13a) in comparison with the
OYE family member PETN reductase. This complementary
activity emphasizes the potential value of NtDBR as an
industrial biocatalyst, in addition to classical OYE “-ene”
reductases. Unlike OYE’s, NtDBR also benefits from the
absence of a bound flavin cofactor. Flavins can be oxidized by
molecular oxygen at the expense of alkene reduction.30 This
results in an overall reduction in reaction rate and generates the
reactive oxygen species (ROS) hydrogen peroxide and
superoxide,19a,30 the presence of which has been shown to
sometimes influence product enantiopurity.31
We have also presented the X-ray crystal structures of the
holo-, binary (NADP(H)-bound), and ternary [NADP+ and 4-
hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR com-
plexes. The use of high-resolution structural information for the
identification of individual residues/interactions, important in
substrate binding and catalysis, is crucial for the generation of
new biocatalysts with improved characteristics. Previously, we
have achieved notable success in redefining the reactivities,
stereochemistries, and specificities of the OYE family member
PETN reductase using a robotic high-throughput kinetic
screening of enzyme mutant libraries.20,32 Although the ternary
complex here shows the substrate bound in a conformation that
is likely to be inhibitory, the position of HMHCA still provides
information to aid the determination of which residues are
likely to be involved in forming the alkene substrate binding
pocket.
Structural and specificity data will be used to drive future
structure-driven high-throughput site-saturated mutagenesis
studies to further expand the biocatalytic potential of this
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(15) Leslie, A. G. W. Recent changes to the MOSFLM package for
processing film and image plate data. Joint CCP4 & ESRF-EACBM
Newsletter on Protein Crystallography; 1992, 26
(16) Collaborative Computational Project Number 4. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760−763.
(17) Perrakis, A.; Morris, R.; Lamzin, V. S. Nat. Struct. Biol. 1999, 6,
458−63.
(18) Krissinel, E.; Henrick, K. J. Mol. Biol. 2007, 372, 774−797.
(19) (a) Fryszkowska, A.; Toogood, H.; Sakuma, M.; Gardiner, J. M.;
Stephens, G. M.; Scrutton, N. S. Adv. Synth. Catal. 2009, 351, 2976−
2990. (b) Toogood, H. S.; Fryszkowska, A.; Hare, V.; Fisher, K.;
Roujeinikova, A.; Leys, D.; Gardiner, J. M.; Stephens, G. M.; Scrutton,
N. S. Adv. Synth. Catal. 2008, 350, 2789−2803.
(20) Hulley, M. E.; Toogood, H. S.; Fryszkowska, A.; Mansell, D.;
Stephens, G. M.; Gardiner, J. M.; Scrutton, N. S. ChemBioChem 2010,
11, 2433−2447.
enzyme. Residues to be targeted include those that line the
substrate binding pocket, although key conserved NAD(P)H-
binding residues will be avoided. Residues further afield, such as
ones involved in the overall protein fold and stability, may be
targeted to allow larger substrates to be accommodated in the
active site. In silico substrate binding studies could also drive
site-directed mutagenesis studies, which may allow the likely
role(s) of specific residues to be determined.
ASSOCIATED CONTENT
■
S
* Supporting Information
Discussion of additional details of the 3D X-ray crystal
structures of DBR. This material is available free of charge
via the Internet at http://pubs.acs.org.
(21) Dick, R. A.; Kensler, T. W. J. Biol. Chem. 2004, 279, 17269−77.
(22) Mano, J.; Torii, Y.; Hayashi, S.; Takimoto, K.; Matsui, K.;
Nakamura, K.; Inze, D.; Babiychuk, E.; Kushnir, S.; Asada, K. Plant Cell
Physiol. 2002, 43, 1445−1455.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +44 161 3065152. Fax: +44 161 3068918. E-mail:
nigel.scrutton@manchester.ac.uk.
(23) Adalbjornsson, B. V.; Toogood, H. S.; Fryszkowska, A.; Pudney,
C. R.; Jowitt, T.; Leys, D.; Scrutton, N. S. ChemBioChem 2010, 11,
197−207.
Author Contributions
(24) Persson, B.; Hedlund, J.; Jornvall, H. Cell. Mol. Life Sci. 2008, 65,
3879−3894.
§These authors contributed equally.
(25) Holm, L.; Park, J. Bioinformatics 2000, 16, 566−567.
(26) Ohta, H.; Kobayashi, N.; Ozaki, K. J. Org. Chem. 1989, 54,
1802−1804.
Notes
The authors declare no competing financial interest.
(27) Fryszkowska, A.; Fisher, K.; Gardiner, J. M.; Stephens, G. M. J.
Org. Chem. 2008, 73, 4295−4298.
ACKNOWLEDGMENTS
■
(28) Shimoda, K.; Izumi, S.; Hirata, T. Bull. Chem. Soc. Jpn. 2002, 75,
813−816.
This work was funded by the UK Biotechnology and Biological
Sciences Research Council (BBSRC; BB/E010717/1). N.S.S. is
a Royal Society Wolfson Merit Award holder and an
Engineering and Physical Sciences Research Council
(EPSRC; EP/J020192/1) Established Career Fellow.
(29) Bougioukou, D. J.; Stewart, J. D. J. Am. Chem. Soc. 2008, 130,
7655−7658.
(30) Williams, R. E.; Bruce, N. C. Microbiology 2002, 148, 1607−
1614.
(31) Fryszkowska, A.; Toogood, H. S.; Mansell, D.; Stephens, G.;
Gardiner, J. M.; Scrutton, N. S. FEBS J. 2012, 279, 4160−4171.
(32) (a) Fryszkowska, A.; Toogood, H.; Sakuma, M.; Stephens, G.
M.; Gardiner, J. M.; Scrutton, N. S. Catal. Sci. Technol. 2011, 1, 948−
957. (b) Toogood, H. S.; Fryszkowska, A.; Hulley, M.; Sakuma, M.;
Mansell, D.; Stephens, G. M.; Gardiner, J. M.; Scrutton, N. S.
ChemBioChem 2011, 12, 738−749.
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