Heterologous expression and characterization of the ene-reductases from Deinococcus radiodurans and Ralstonia metallidurans

Article abstract of DOI:10.1016/j.molcatb.2013.10.020

The Old Yellow Enzyme (OYE) homologues or ene-reductases (ER) from Deinococcus radiodurans (DrER) and Ralstonia metallidurans (RmER) were cloned and characterized. Sequence and phylogenetic analysis revealed both these enzymes to belong to the YqjM-like or "thermophilic-like" group of OYEs, both sharing more than 60% sequence similarity to the ER from Thermus scotoductus. This group of OYEs is characterized by a conserved cysteine residue modulating the redox potential of the flavin cofactor as well as a conserved tyrosine residue located at the N-terminus region involved in binding certain ligands. The genes were recombinantly expressed in Escherichia coli as functional soluble proteins. Both ERs have monomer molecular weights of approximately 40 kDa, with DrER a homodimer in solution and RmER a monomer. DrER and RmER are optimally active at pH 7-7.5 at 30 C and 35 C respectively. Although the enzymes showed comparable affinities towards the ubiquitous ER substrate 2-cyclohexenone, the specific activity and catalytic efficiency of DrER were more than twice those observed for RmER. Similar to other members of this subclass of ERs, no conversion was detected with cyclic Cβ substituted enones, and only DrER was able to convert citral. Both DrER and RmER were highly active at reducing N-phenyl substituted maleimides. The selectivity of the ERs was assessed using both the isomers of carvone, which were converted with high diastereomeric excesses. Ketoisophorone and 2-methylcyclopentenone were converted to their (R)- and (S)-enantiomeric products respectively. Finally, a light-driven cofactor regeneration system was used to drive enzymatic reduction in the absence of NAD(P)H.

Full text of DOI:10.1016/j.molcatb.2013.10.020

Journal of Molecular Catalysis B: Enzymatic 99 (2014) 89–95
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Heterologous expression and characterization of the ene-reductases
from Deinococcus radiodurans and Ralstonia metallidurans
S. Litthauera, S. Gargiulob, E. van Heerdena, F. Hollmannb, D.J. Oppermana,∗
a University of the Free State, Department of Biotechnology, 205 Nelson Mandela Drive, Bloemfontein 9300, South Africa
b Delft University of Technology, Department of Biotechnology, Julianalaan 136, 2628BL Delft, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 August 2013
Received in revised form 3 October 2013
Accepted 25 October 2013
Available online 1 November 2013
The Old Yellow Enzyme (OYE) homologues or ene-reductases (ER) from Deinococcus radiodurans (DrER)
and Ralstonia metallidurans (RmER) were cloned and characterized. Sequence and phylogenetic analy-
sis revealed both these enzymes to belong to the YqjM-like or “thermophilic-like” group of OYEs, both
sharing more than 60% sequence similarity to the ER from Thermus scotoductus. This group of OYEs is
characterized by a conserved cysteine residue modulating the redox potential of the flavin cofactor as
well as a conserved tyrosine residue located at the N-terminus region involved in binding certain ligands.
The genes were recombinantly expressed in Escherichia coli as functional soluble proteins. Both ERs have
monomer molecular weights of approximately 40 kDa, with DrER a homodimer in solution and RmER
a monomer. DrER and RmER are optimally active at pH 7–7.5 at 30 ◦C and 35 ◦C respectively. Although
the enzymes showed comparable affinities towards the ubiquitous ER substrate 2-cyclohexenone, the
specific activity and catalytic efficiency of DrER were more than twice those observed for RmER. Sim-
ilar to other members of this subclass of ERs, no conversion was detected with cyclic C␤ substituted
enones, and only DrER was able to convert citral. Both DrER and RmER were highly active at reducing N-
phenyl substituted maleimides. The selectivity of the ERs was assessed using both the isomers of carvone,
which were converted with high diastereomeric excesses. Ketoisophorone and 2-methylcyclopentenone
were converted to their (R)- and (S)-enantiomeric products respectively. Finally, a light-driven cofactor
regeneration system was used to drive enzymatic reduction in the absence of NAD(P)H.
Keywords:
Old Yellow Enzyme
Ene-reductase
␣,␤-Unsaturated carbonyl
Cofactor regeneration
Flavin oxidoreductase
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
hydride transfer to the flavin mononucleotide (FMN) cofactor, fol-
lowed by the subsequent binding of the substrate and the concerted
The Old Yellow Enzyme (OYE) family or ene-reductases (ER)
is a ubiquitous group of flavoproteins that catalyze the asym-
metric reduction of activated C C bonds of a wide variety of
␣/␤-unsaturated carbonyl compounds. First described as a yellow
enzyme involved in the oxidation of NADPH by molecular oxygen,
Old Yellow Enzyme was the first flavin-containing enzyme charac-
terized and played an important role by serving as model enzyme in
studies aimed at understanding the role of flavin cofactors in pro-
teins [1,2]. Twenty years after Stott and co-workers [3] reported
2-cyclohexenone as an alternative electron acceptor for OYE1 from
Saccharomyces pastorianus (formerly carlsbergensis), the substrate
range of OYE has broadened to now include a variety of cyclic and
acyclic enones and enals (for recent reviews see [4,5]).
transfer of the hydride from the reduced FMN to the C␤ of the alkene
and a proton to the C␣ [6]. This proton is donated from an active-site
tyrosine, or can be directly derived from the solvent [7,8]. Impor-
tantly, the oxidative half-reaction results in a net trans addition
which occurs with absolute stereospecificity. OYEs have therefore
become attractive biocatalysts due to their ability to perform trans-
hydrogenation [9] as an alternative to current chiral organometallic
catalysts [10] for enantioselective hydrogenation. This asymmetric
reduction yields enantiopure chiral building blocks, important in
the synthesis of various fine chemicals and pharmaceuticals [11].
While the physiological function of these enzymes remains elusive,
additional substrates and activities have been identified, including
the reduction of ␣,␤-unsaturated dicarboxylic acids and dimethyl
esters [12], maleimides [13,14], nitroalkenes [15,16], steroids [17]
as well as the reductive denitration of nitrate esters and nitroaro-
matics [18–20].
The reduction of these activated alkenes occurs through a ping-
pong bi–bi mechanism whereby both the oxidant and reductant
bind in the same active site sequentially. NAD(P)H is oxidized by
The list of OYEs has also been growing steadily to now include
OYE homologous characterized, both biocatalytically and struc-
turally, from yeast [21], plants [22,23], bacteria [24–27] and
recently thermophilic bacteria [28–30], allowing biocatalysis at
∗
Corresponding author. Tel.: +27 51 401 2714; fax: +27 51 401 9376.
E-mail address: opperdj@ufs.ac.za (D.J. Opperman).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.10.020

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S. Litthauer et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 89–95
higher temperatures. Although the overall structure and mecha-
nism of these OYE homologues are conserved, slight changes within
the catalytic site architecture of these enzymes are yielding dif-
ferent stereo-specificities. These stereopreferences have recently
been structurally investigated to predict biocatalytic properties
based on sequence data [31]. In addition, directed evolution stud-
ies to broaden substrate scope as well as to engineer reversed
stereochemical outcomes through different substrate binding ori-
entations [32–34] are also elucidating critical factors in substrate
specificity and selectivity, in addition to expanding our catalytic
toolbox.
One of the subclasses of ERs, the so-called YqjM-like or
“thermophilic-like” OYEs is dominated by the recently described
ERs from thermophiles [5]. Only two mesophilic counterparts are
found within this subclass, YqjM from Bacillus subtilis [26] and XenA
from Pseudomonas putida [25]. Here we report the cloning, het-
erologous expression and characterization of two new mesophilic
ene-reductases from Deinococcus radiodurans and Ralstonia metal-
lidurans, belonging to this underrepresented subclass. Finally, we
investigate the use of a light-driven co-factor regeneration system
with both ERs for the photobiocatalytic reduction of C C bonds.
The pET28-ene-reductase constructs were transformed into E. coli
BL21(DE3) competent cells (Lucigen) and selected on LB-plates con-
taining 30 ␮g/mL kanamycin.
2.3. Heterologous expression and protein purification
Expression of the ene-reductases was performed using auto-
induction media (ZYP5052 medium; [35]) at 25 ◦C (200 rpm) for
24 h. Cells were harvested through centrifugation (5000×g, 15 min)
and washed twice and resuspended (1 g wet weight cells in 10 mL
buffer) in 20 mM MOPS-NaOH (pH 7.4) and 0.1 M NaCl buffer. Cells
were broken using a One Shot Cell Disruption system (Constant
Systems Ltd) at 30 KPSI. Unbroken cells and debris were removed
through centrifugation (5000×g, 20 min). The soluble fraction
(cytoplasm) was obtained through ultracentrifugation (100,000×g
for 90 min).
Recombinant N-terminally His6-tagged proteins were purified
by immobilized metal-affinity chromatography (IMAC) and size-
exclusion chromatography. The soluble fractions were loaded onto
HisTrap FF columns (5 mL, GE Healthcare) equilibrated with 20 mM
MOPS-NaOH (pH 7.4) containing imidazole (40 mM) and NaCl
(0.5 M). Unbound proteins were removed by washing (5 mL/min)
with the same buffer. Recombinant ERs were then eluted in the
same buffer with use of an increasing linear gradient (100 mL) of
imidazole up to 0.5 M. Fractions containing the characteristic yel-
low colour were pooled for subsequent purification. All protein
samples were incubated with excess FMN before size-exclusion
chromatography (SEC). Samples were concentrated to approxi-
mately 3 mL by ultrafiltration (30 kDa MWCO, Millipore) and loaded
onto a Sephacryl S-200HR columns (65 × 2.5 cm, Sigma) equili-
brated with 20 mM MOPS-NaOH (pH 7.4) and 0.1 M NaCl. Proteins
were eluted with the same buffer at a flow speed of 1 mL/min.
Alternatively, samples from the Ni-affinity chromatography step
were desalted using PD-10 desalting columns (GE Healthcare) into
20 mM MOPS-NaOH and 0.1 M NaCl (pH 7.4). Protein concentra-
tions were determined with the BCA protein assay kit (Pierce) with
bovine serum albumin (BSA) as standard. Enzyme purity was eval-
uated using SDS-PAGE [36] stained with Coomassie brilliant blue
R-250. PageRuler protein ladder (Fermentas) was used as molecular
mass markers. Quaternary structures of the ERs were determined
by SEC using Gel Filtration standards (Bio-Rad) and BSA.
2. Experimental
2.1. Bacterial strains and culture conditions
Deinococcus radiodurans type strain and Ralstonia (Cupriavidus)
metallidurans strain CH34 was obtained from the Leibniz Institut
DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkul-
turen GmbH (DSMZ). D. radiodurans was cultured in nutrient broth
consisting of 5 g/L peptone and 3 g/L meat extract (pH 7, 200 rpm,
30 ◦C) and R. metallidurans was cultured in Corynebacterium broth
consisting of 10 g/L peptone, 5 g/L yeast extract, 5 g/L glucose and
5 g/L NaCl (pH 7.2, 200 rpm, 35 ◦C). E. coli strains were grown in
Luria–Bertani (LB) medium at 37 ◦C with shaking (200 rpm).
2.2. Cloning and heterologous expression of the ERs
Genomic DNA (gDNA) was isolated from the Deinococcus and
Ralstonia strains using Aquapure Genomic DNA kit (Bio-Rad) as
per manufacturer’s instructions. The complete open reading frames
(ORFs) were PCR amplified using the Expand high-fidelity PCR sys-
tem (Roche) with oligonucleotides DR F1 Nde (5ꢀ CAT ATG ACC
GTG TCT TCC GCC GCT GCA CC 3ꢀ) and DR R1 Eco (5ꢀ GAA TTC TTA
CCA CCC CGC CCG CGC GTA CTG 3ꢀ) for the ER from Deinococcus
and RM F1 Nde (5ꢀ CAT ATG CCT CAT CTC TTC GAT CCG TAC C 3ꢀ)
and RM R1 Eco (5ꢀ GAA TTC TCA ACG CTG GCC GAA GTG CGC GT
3ꢀ) for the ER from Ralstonia. Primers contained NdeI and EcoRI
restriction sites (underlined) for directional cloning and incorpora-
tion of a N-terminal hexahistidine-tag and thrombin cleavage site
from the pET vector. Reaction mixtures (50 ␮L) consisted of 10×
Expand high-fidelity buffer with 15 mM MgCl2 (5 ␮L), deoxynucle-
oside triphosphates (0.2 mM each), Expand high-fidelity enzyme
mix (3.5 U), 50 ng of gDNA, and 0.2 ␮M of both the forward and
reverse primers. PCR conditions consisted of an initial denatur-
ing step at 95 ◦C for 5 min, followed by 25 cycles of denaturing
at 95 ◦C (30 s), annealing at 62 ◦C (40 s), and elongation at 72 ◦C
(1.5 min), with a final extension at 72 ◦C for 10 min. Purified PCR
products (Biospin gel extraction kit, BioFlux) were ligated into
pGEM-T Easy (Promega) and transformed into E. coli TOP10 com-
petent cells (Invitrogen) and selected for on LB-plates containing
100 ␮g/mL ampicillin. Plasmid DNA was isolated (Biospin plasmid
DNA extraction kit, BioFlux) and verified by DNA sequencing. For
expression of the ene-reductases, the ORFs were sub-cloned into
pET28b(+) (Novagen) using the NdeI and EcoRI restriction sites.
2.4. Enzyme assays
Steady-state kinetics of the purified ene-reductases were per-
formed by measuring the rate of NAD(P)H oxidation at 340 nm
(Cary 300Bio UV/Vis spectrophotometer) with use of an extinc-
tion coefficient of 6.22/mM/cm. Assays were performed in 1 mL
reaction volumes containing NADPH (0.3 mM), 2-cyclohexen-1-
one and the purified protein [RmER = 10–13 ␮g (0.24–0.31 nmol);
DrER = 5–6 ␮g (0.12–0.15 nmol)]. Reactions were performed in
20 mM MOPS-NaOH (pH 7.4) containing 0.1 M NaCl at 30 ◦C. Assays
were performed under aerobic conditions and NADPH oxidation by
the enzyme due to molecular oxygen was measured independently
and subtracted from the total oxidation rates with substrates.
Biotransformation for substrate scope and selectivity analy-
sis were performed in 1 mL reaction volumes consisting of 2 mM
NADH and 1 mM substrate, purified enzyme (20 ␮g) in 20 mM
MOPS-NaOH (pH 7.4) with 0.1 M NaCl buffer at 30 ◦C. Conver-
sions were determined after 5 h of incubation. For GC–MS analysis,
reaction mixtures were extracted using an equal volume of ethyl
acetate and samples separated on a FactorFour VF-5ms column
(30 m × 0.25 mm × 0.25 ␮m, Varian). Chiral separation of the reduc-
tion products of R- and S-carvone, 2-methylcyclopentenone and
ketoisophorone were performed on a Astec Chiraldex G-TA col-
umn (30 m × 0.25 mm × 0.25 ␮m, Sigma–Aldrich) and compared to

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91
reference activities [37] or the MS fragmentation of diastereomers
[38]. For HPLC analysis, reactions were stopped by the addition of
10 ␮L of concentrated HCl and samples separated on a Jupiter 5 ␮m
C18 300 A column (250 mm × 4.6 mm, Phenomenex).
all other “classical” OYEs have, similar to OYE1, a corresponding
conserved threonine (Fig. 1). This cysteine has been implicated in
modulating the reduction potential of the flavin [43]. This group
also has a conserved Tyr (Tyr27) near the N-terminus which has
been shown to participate in the binding of certain ligands [26,29].
The Arg-finger (Arg347) observed in TsER and YqjM appears to be
conserved, with only XenA having a tryptophan residue from the
neighbouring monomer extending into the catalytic site. The mul-
tiple alignments also revealed DrER and RmER to vary notably in
the length of their N- and C-termini. DrER and RmER contain more
than 12 additional residues on their N- and C-termini respectively,
relative to each other as well as other members of this group.
˚
2.5. Co-factor regeneration
The efficacy of a light-driven cofactor regeneration system in
the reduction of 2-cyclohexenone using both DrER and RmER was
investigated. Reactions were performed on a 10 mL scale in closed
glass Hungate tubes and consisted of 5 mM EDTA and substrate,
0.3 mM FMN (0.05 mM for prochiral substrates) and 0.1 mg/mL
(2.4 ␮M) enzyme in 20 mM MOPS-NaOH (pH 7.4) with 0.1 M NaCl
buffer at 30 ◦C. Oxygen was removed from the reaction mixture by
passing N2 gas through the reaction mixture for 10 min before the
enzyme was added. The reaction mixture was illuminated for 24 h
with samples withdrawn under a N2 gas stream and extracted with
one volume equivalent of ethyl acetate for GC–MS analysis. Light
was provided as cold light by a KL 1500 compact (Leica) fitted with
a 150 W bulb (5 cm distance).
3.2. Protein expression, purification and physicochemical
characterization
Both DrER and RmER were expressed as soluble N-terminally
histidine-tagged variants with apparent monomer molecular
weights of approximately 38 kDa (Fig. 2). The ERs were purified
to homogeneity with typical yields between 160 and 200 mg/L
E. coli culture using IMAC and size-exclusion chromatography
(SEC; Fig. 3). SEC also showed the DrER to exhibit a homodimeric
quaternary structure similar to TsER, whereas RmER occurred as
monomers in solution, possibly due to its extended C-terminus or
possibly by the interference by the hexahistidine tag and thrombin-
cleavage site from the pET vector [44]. Although sequence based
analysis shows the Arg-finger to be conserved in RmER, its
monomeric structure suggests a more open catalytic site without
the Arg-finger from an adjacent monomer. The thermophilic-like
ERs tend to vary greatly in their quaternary structure, with TsER
occurring as dimers in solution [29] and TOYE from Thermoanaer-
obacter even occurring in multiple oligomeric states (tetramers to
dodecamers) in vitro [30].
2.6. Phylogenetic analysis
Old Yellow Enzyme homologue sequences were retrieved from
the National Centre for Biotechnology Information (NCBI). Mul-
tiple sequence alignments of the amino acid sequences were
performed using the MUSCLE EBI web tool (http://www.ebi.ac.uk/
Tools/msa/muscle) with the default parameters [39]. The best
amino acid substitution model was estimated for tree building
using the MEGA 5 software [40]. The Whelan and Goldman (WAG)
model [41] was selected with a discrete Gamma distribution with
5 rate categories and by assuming that a certain fraction of sites
are evolutionary invariable. An un-rooted maximum likelihood tree
was constructed using MEGA 5. The phylogenetic tree was inferred
using nearest-neighbour-interchange (NNI) with bootstrap sup-
port for individual nodes calculated on 500 replicates.
The purified proteins had an intense yellow colour due to the
bound flavin (FMN) and physicochemical characterization revealed
that both enzymes function optimally near the optimal growth
temperature and pH of their original bacterial sources. The opti-
mum pH and temperature conditions for both the DrER and RmER
was determined to be 7–7.5 at 30 ◦C and 35 ◦C respectively.
3. Results and discussion
3.1. Cloning of the ene-reductase
3.3. Substrate scope and kinetic characterization
Similarity searches using BLASTp of whole-genome sequences
identified two open reading frames (ORFs) encoding for typical
orthologues of Old Yellow Enzyme/ene-reductases in the D. radio-
durans genome (DrER and DrER2) and only one in R. metallidurans
(RmER). The protein sequences of the DrERs share only 37% sim-
ilarity with each other, with DrER2 also showing an unusually
long C-terminus, more than 50 residues longer than DrER. The
1113 bp and 1116 bp ORFs encoding the putative ene-reductases,
RmER and DrER, were successfully amplified from genomic DNA,
whereas no PCR product could be obtained for DrER2. Both these
ene-reductases showed the highest similarity at protein level
to the ER characterized from Thermus scotoductus (TsER) [29,42]
with 48% and 46% identity (61% and 60% similarity) for DrER and
RmER respectively. Multiple alignments with previously cloned
and characterized ene-reductases and phylogenetic analysis (Fig. 1)
showed both these putative ERs to group with the YqjM-type (or
“thermophilic-like”) ERs that also include XenA from Pseudomonas
putida [25] as well as the OYEs from the thermophiles T. scotoductus,
Thermoanaerobacter pseudethanolicus (TOYE; [30]) and Geobacillus
kaustophilus (GkOYE; [28]).
Both enzymes catalyzed the reduction of 2-cyclohexen-1-one
through the oxidation of NADPH or NADH, with a preference for
NADPH as electron donor (Fig. 4). Substrate inhibition was observed
when NADH was used as electron donor. As ERs function through
compulsory ordered substrate binding and product release through
a ping-pong bi–bi mechanism, a lower binding affinity for NADH
can cause 2-cyclohexenone to act as a competitive inhibitor at high
concentrations for the reductive-half reaction, effectively binding
before the enzyme is reduced (dead-end inhibition). Saturation
kinetics with NADPH (Table 1) revealed similar affinity (Km) of both
enzymes for 2-cyclohexenone, although DrER had a higher specific
activitycomparedto RmER, makingthe catalyticefficiency(kcat/Km
)
of DrER almost three times higher than that observed for RmER
towards cyclohexenone.
To explore the substrate scope and selectivity, a range of
potential substrates were investigated (Table 2). Biotransformation
reactions were performed using purified enzymes and NADH as
cofactor. Cyclic enones with methyl substitutions on the C␤ posi-
tion (1d, 1e) were not reduced by either DrER or RmER, similar
to other members of this group such as YqjM and TsER. The only
enal tested, citral (3), has a methyl group on the C␤ and reactiv-
ity was only detected for DrER. 2-Methylcyclopentenone (1c) and
ketoisophorone (1f) were reduced by both DrER and RmER, albeit
marginally, except for DrER which reduced approximately 75%
This subclass of ERs all has a conserved histidine pair (His175
and His172, TsER numbering) for the hydrogen bonding of the sub-
strate’s carbonyl oxygen, as well as a conserved proton donating
tyrosine for the oxidative half reaction (Tyr177). The YqjM-like
group of ERs has a conserved cysteine (Cys25) residue, whereas

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S. Litthauer et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 89–95
Fig. 1. (a) Phylogenetic relationship of DrER and RmER with previously cloned and characterized ene-reductases/OYEs through a bootstrap consensus un-rooted maximum-
likelihood (ML) tree inferred from the WAG + I + G model. (b) Catalytic site of TsER with p-hydroxybenzaldehyde bound (PDB ID: 3GHJ) above the flavin mononucleotide
(FMN) showing the conserved amino acids involved in catalysis and substrate binding. (c) Multiple sequence alignment of the ERs used for comparison with NCBI accession
numbers given in parentheses. OYE2 and 3 Saccharomyces cerevisiae (AAA83386, AAA64522), OYE1 Saccharomyces pastorianus (carlsbergensis; Q02899), KYE1 Kluyveromyces
lactis (AAA98815), CaMa Candida macedoniensis (BAD24850), OPR1 and 3 Solanum lycopersicum (AJ242551, NP 001233873), SYE1 Shewanella oneidensis (AAN55488), NCR
Zymomonas mobilis (Q5NLA1), XenB Pseudomonas fluorescens (AF154062), MorR Pseudomonas putdia (AAC43569), Yers-ER Yersinia bercovieri (WP 005270358), PETNR Entero-
bacter cloacae (U68759), NemA Escherichia coli (BAA13186), DrER Deinococcus radiodurans (NP 295913), XenA Pseudomonas putida (AF154061), RmER Ralstonia metallidurans
(YP 586990), TsER Thermus scotoductus (AM902709), TOYE Thermoanaerobacter pseudethanolicus (YP 001664021), YqjM Bacillus subtilis (BAA12619), GkOYE Geobacillus
kaustophilus (YP 148185). YqjM-like or “thermophilic-like” ERs are shown by shading in light grey. Dark grey shading indicates the catalytically important conserved
amino acids. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)
Fig. 3. Elution profile from size-exclusion chromatography of DrER (᭹) and
RmER (ꢁ). Reference standards are shown as triangles with molecular weights
in kDA. Inset: calibration curve, bovine ␥-globulin (158 kDa), bovine serum
albumin (66.5 kDa), chicken ovalbumin (44 kDa) and horse myoglobin (17 kDa).
Kav = (Ve − V0)/(Vt − V0).
Fig. 2. SDS-PAGE analysis of the expression and purification of RmER and DrER. 1:
Molecular weight marker, 2: soluble extract of E. coli, 3: soluble extract of E. coli
expressing RmER, 4: purified RmER, 6: soluble extract of E. coli expressing DrER, 7:
purified DrER.

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93
Scheme 1. Schematic representation of the light driven cofactor regeneration path-
way for the reduction of conjugated C C double bonds. E-FMN represents the
enzyme bound flavin group.
DrER. Moreover, multiple binding conformations have been shown
to influence enantiopurity, and in the case of the reduction of 2-
methylcyclopentenone, a “flipped” binding orientation [32] relative
to cyclohexenone derivatives accounts for the reversal of the ste-
reochemical outcome, yielding the S-enantiomer.
3.4. Cofactor regeneration
One of the major drawbacks still in the application of ERs is the
requirement for expensive co-factors. Most researchers therefore
use whole-cell systems, which often give low yields and enantios-
electivies due to the interfering native ene-reductases or alcohol
dehydrogenases [9,46]. Various co-factor regeneration systems are
therefore being employed to overcome this limitation not only for
ERs but also redox enzymes in general [47].
Enzymatic co-factor regeneration systems such as glucose
dehydrogenase, glucose-6-phosphate dehydrogenase and formate
dehdrogenase have been shown to affect yield and enantiopurity
of products [48–50]. An alternative to these conventional enzy-
matic cofactor systems is the use of light-driven direct regeneration
[51,52]. Previous attempts to use the light-driven cofactor regen-
eration system with TsER were problematic due to the fact that
EDTA, which is used as sacrificial electron donor, binds calcium and
magnesium which TsER requires for activity. We therefore tested
DrER and RmER for their applicability with this system using 2-
cyclohexenone (1a) as model substrate (Scheme 1).
Complete conversion of 5 mM 2-cyclohexanone was obtained
with both DrER and RmER using the light-driven cofactor recycling
(Fig. 5). Although initial kinetic characterization of the two
ERs showed the DrER to have a higher specific rate with 2-
cyclohexenone using NADPH as electron donor, surprisingly RmER
was able to catalyze the complete conversion within 5 h, show-
ing nearly twice the reaction rate observed with DrER. The effect
Fig. 4. Steady-state kinetics of DrER (᭹) and RmER (ꢁ) using varying concentrations
of 2-cyclohexenone and 0.3 mM NADPH (a) or NADH (b). Error bars indicate standard
deviation.
of the ketoisophorone within 5 h. The dimethyl ␣,␤-unsaturated
esters, dimethylfumarate (2a) and dimethylmaleate (2b), also
served as substrates for both DrER and RmER, with the cis con-
figuration the preferred substrate in both cases. Structurally more
demanding substrates such as the isomers of carvone (4), proved
to be converted by both DrER and RmER. The (R)-(−)-carvone
(4a) was slightly preferred by both DrER and RmER. Apart from
2-cyclohexenone, 2-methyl-N-phenylmaleimide (5a) and its un-
substituted counterpart N-phenylmaleimide (5b), were the only
substrates being completely converted after 5 h by both DrER and
RmER.
The enantioselectivity of the ERs was evaluated using
the isomers of carvone (4), ketoisophorone (1f), and 2-
methylcyclopentenone (1c). Both ERs showed high enantiose-
lectivity, forming 2R,5S-dihydrocarvone (>93% de) and 2R,5R-
dihydrocarvone (>95% de) from R-(−)-carvone (4a) and S-(+)-
carvone (4b) respectively. Similar to other members of this
subgroup of ERs, ketoisophorone (1f) and 2-methylcyclopentenone
(1c) were converted to their (R)- and (S)-enantiomeric products
respectively. The low optical purity of (6R)-levodione (product
of 1f) has been attributed to the racemization of the product in
aqueous solutions [45], likewise we noted a decrease in optical
purity to approximately 90% ee after 24 h with both RmER and
Fig. 5. Time course of 2-cylcohexenone reduction using DrER (᭹) and RmER (ꢁ)
with the light driven cofactor regeneration system.

94
S. Litthauer et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 89–95
Table 1
Catalytic parameters of purified DrER and RmER towards 2-cyclohexenone with NADPH as electron donor.
Vmax (␮mol min−1 mg−1
)
Km (mM)
kcat (s−1
)
kcat/Km (mM−1 s−1
)
DrER
RmER
14.77 0.60
5.49 0.35
4.92 0.53
5.51 0.88
10.28
3.87
2.09
0.70
Conditions: NADPH = 0.3 mM, RmER (13 ␮g/ml) and DrER (6 ␮g/ml), 20 mM MOPS-NaOH (pH 7.4) containing 0.1 M NaCl at 30 ◦C.
Table 2
Substrate scope and selectivity of DrER and RmER.
Substrate
Conversiona (%)
Selectivity (ee/de) (%)
DrER
DrER
RmER
RmER
O
1a
>99
>99
O
O
O
1b
1c
73.5
8.8
62.8
6.5
58% (S)
>99% (S)
1d
1e
n.c.
n.c.
n.c.
n.c.
O
O
1f
75.3
45.6
93.9
3.0
99% (R)
97% (R)
O
O
O
2a
2b
20.9
52.0
O
O
O
O
O
O
O
3
8.5
n.c.
n.d.
O
O
4a
4b
99.0
92.1
83.1
56.0
95% (2R,5R)
93% (2R,5S)
96% (2R,5R)
93% (2R,5S)
O
N
O
O
5a
5b
>99
>99
>99
>99
n.d.
n.d.
O
N
n.c. No conversion detected after 5 h conversion.
n.d. Not determined.
a
Percentage conversion determined by product to substrate ratios of GC analysis. All substrate concentrations were 1 mM, 2 mM NADH, 20 ␮g/mL ER, 5 h at 30 ◦C in 20 mM
MOPS pH 7.4 with 0.1 M NaCl.

S. Litthauer et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 89–95
95
Table 3
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lidurans, both belonging to the YqjM-like subclass of OYEs were
cloned, successfully expressed in E. coli and purified. Testing
of different substrate classes revealed slight differences in the
two ERs catalytic preferences and enantioselectivies. For most of
the substrates tested, the specific activity of DrER was greater
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Acknowledgements
This work was supported by the National Research Foundation
South Africa and the Oppenheimer Memorial Trust. The authors
thank Maarten Gorseling, Remco van Oosten (Delft University of
Technology, NL) and Sarel Marais (University of the Free State, SA)
for GC and HPLC analysis.
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