Enhanced Ene-Reductase Activity through Alteration of Artificial Nicotinamide Cofactor Substituents

Article abstract of DOI:10.1002/cctc.201501230

The reduction of activated C=C double bonds is an important reaction in synthetic chemistry owing to the potential formation of up to two new stereogenic centers. Artificial nicotinamide cofactors were recently presented as alternative suppliers of hydride equivalents needed for alkene reduction. To study the effect of cofactors on the reduction of activated alkenes, a set of N-substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthetic cofactors on enzyme activity of four ene reductases are outlined in this study, where the cofactor mimic with an N-substituted 4-hydroxy-phenyl residue led to a sixfold higher v_max relative to the natural cofactor NADH. Artificial nicotinamide cofactor substituents: A set of N-substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthesized cofactors on the enzyme activity of four ene reductases are outlined. The cofactor mimic with an N-substituted 4-hydroxy-phenyl residue led to a sixfold higher v_max relative to the natural cofactor NADH.

Full text of DOI:10.1002/cctc.201501230

DOI: 10.1002/cctc.201501230
Full Papers
Enhanced Ene-Reductase Activity through Alteration of
Artificial Nicotinamide Cofactor Substituents
Sebastian A. Lçw,[a] Isabell M. Lçw,[b] Martin J. Weissenborn,[a] and Bernhard Hauer*[a]
The reduction of activated C=C double bonds is an important
reaction in synthetic chemistry owing to the potential forma-
tion of up to two new stereogenic centers. Artificial nicotina-
mide cofactors were recently presented as alternative suppliers
of hydride equivalents needed for alkene reduction. To study
the effect of cofactors on the reduction of activated alkenes,
a set of N-substituted synthetic nicotinamide cofactors with
differing oxidation potentials were synthesized and their elec-
trochemical and kinetic behavior was studied. The effects of
the synthetic cofactors on enzyme activity of four ene reduc-
tases are outlined in this study, where the cofactor mimic with
an N-substituted 4-hydroxy-phenyl residue led to a sixfold
higher vmax relative to the natural cofactor NADH.
Introduction
Ene reductases are interesting alternatives for organic chemists
to chemical methods for the enantioselective reduction of acti-
vated triple or double bonds.[1,2] Ene reductases have proved
to be excellent catalysts in terms of selectivity,[3] stability,[4] and
tolerance towards enzyme engineering.[5] They are able to ste-
reoselectively reduce double bonds in conjugation with alde-
hydes,[6] ketones,[7] nitroalkenes,[8] maleimids,[9] dicarboxylic
acids,[10] esters,[11] and nitriles.[12] Ene reductases rely on a two-
step ping-pong-bi-bi mechanism (Scheme 1). The flavin mono-
nucleotide (FMN)-containing active site first binds the nicotina-
mide cofactor NAD(P)H, which reduces the prosthetic flavin. In
a second step, the substrate binds to the active site and a hy-
dride is transferred from the flavin to the b-carbon of the
double bond in a Michael-type addition, leading to ee values
of >99.5%.[13] The modification of the necessary nicotinamide
cofactor reaches back to the 1930s. Karrer and co-workers
identified the nicotinamide moiety of the NADPH as the active,
hydride transferring part of the molecule for ene-reductase-cat-
alyzed reactions.[14] This result implies that the adenine dinucle-
otide residue is mostly utilized for the recognition and posi-
tioning of the cofactor.[15] Previous work described several ene
reductases to be promiscuous with regards to their specificity
towards NADH and NADPH.[4,14,16–19] Owing to this cofactor pro-
miscuity, different architectures of the hydride donor are feasi-
ble alternatives[20,21] as well as employing H2O with a TiO2-
Scheme 1. Reaction scheme of a flavin-mediated reduction of an activated
C=C double bond. The first step is limited by the kinetic constant k1 and
leads to a reduced flavin. The second step is the reduction of substrate with
the kinetic constant k2. If k1 <k2, then the flavin reduction is the rate limiting
step.
based photocatalyst as a hydride donor,[22] and the utilization
of an electrode system with methyl viologen as a mediator.[23]
The alteration of the nicotinamide function has, moreover,
been studied with ene reductases. Recent work revealed two
predominant positions for modification of the nicotinamide
ring.[24] First, the amide function was replaced by other elec-
tron-withdrawing groups such as cyanide and carboxyl.
Second, the adenine dinucleotide moiety was replaced by vari-
ous other aliphatic and benzylic groups. With these simplified
cofactors, conversions using natural NADH could be matched.
In contrast to flavin-containing enzymes, the direct hydride
transfer from the cofactor to the substrate with alcohol dehy-
drogenases was not feasible when cofactor mimics were ap-
plied. This enabled the bio-selection in crude cell lysate, where
competing reduction reactions of alcohol dehydrogenase and
ene reductase take place.[24] As yet, there has not been an ex-
ample of a significantly enhanced enzyme activity with altered
cofactors. By means of kinetic investigations using the ene re-
[a] S. A. Lçw, Dr. M. J. Weissenborn, Prof. Dr. B. Hauer
Institute of Technical Biochemistry
University of Stuttgart
Allmandring 31, 70569 Stuttgart (Germany)
E-mail: bernhard.hauer@itb.uni-stuttgart.de
[b] I. M. Lçw
Institute of Inorganic Chemistry
University of Stuttgart
Pfaffenwaldring 55, 70569 Stuttgart (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201501230.
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ductase, old yellow enzyme 1 (OYE1), the flavin-reducing step
(k1) was identified to be slower than the substrate reduction
(k2).[25] Therefore, a faster flavin reduction should lead to an in-
creased overall reaction rate (Scheme 1). We reasoned that by
varying the electrochemical potential of the utilized cofactor,
an improved k1 might be observed. Aromatic residues attached
to the endogenous nitrogen atom could function as electron-
donating residues and alter the electrochemical properties. In
the present paper, we discuss the effect of cofactor potentials
by substituting the adenine dinucleotide with different aromat-
ic residues and the consequent influence on activity by using
four ene reductases. The electrochemical properties of these
cofactors were measured by cyclic voltammetry. Kinetic experi-
ments revealed a sixfold increase in enzyme activity.
Figure 2. Overlapped cyclic voltammograms of NADH and the synthetic ana-
logs BNAH, PNAH, and HPNAH. Measured in 0.1m Bu4NClO4 solutions in
DMSO with a glassy carbon working electrode, Pt counter electrode, and
Ag/AgCl reference. The ferrocene/ferrocenium (Fc/Fc+) couple served as an
internal reference. Red refers to NADH, blue refers to BNAH, purple refers to
PNAH, and black refers to HPNAH.
Results and Discussion
Cofactor design
The so-far investigated cofactors have in common that only
sp3-hybridized carbon atoms have been attached to the heter-
ocyclic amine. The direct coupling of an aromatic system carry-
ing various substituents to the nicotinamide ring offers many
possibilities to alter the steric and electrochemical behavior of
the cofactor. We synthesized the cofactors 2 and 4 by using
Zincke’s salt (3-carbamoyl-1-(2,4-dinitrophenyl)pyridine-1-ium-
chloride) and named them similarly to the already described 1-
benzyl-1,4-dihydro-nicotinamide (BNAH). Thus, PNAH relates to
1-phenyl-1,4-dihydro-nicotinamide and HPNAH relates to 1-(4-
hydroxyphenyl)-1,4-dihydro-nicotinamide (Figure 1). The al-
ready described BNAH was synthesized as published previously
and used for comparison.[24,26–28]
mesomeric substituent effects, PNAH behaved similarly to
NADH. HPNAH—which carries an additional para-hydroxy
group—showed the lowest oxidation potential. A lower oxida-
tion potential could indicate a higher ability for hydride dona-
tion.[31] BNAH, possessing only an inducing effect from the
methylene group, revealed a higher oxidation potential than
HPNAH and a lower one than NADH. After the first oxidation,
a new process around À1 V was observed, which was assigned
to a decomposition product as it was not observed when start-
ing the measurements with the reduction step. PNAH and
HPNAH showed a difference of 0.2 V between their oxidation
potentials, which was highly suitable for studying the influence
of the electrochemical potential of the cofactor. The difference
between our values and those in the literature is due to an al-
tered experimental setup.[32–35]
Enzyme screening
As the altered electronic properties of the cofactor derivatives
BNAH, PNAH, and HPNAH could influence their interactions
with the prosthetic flavin group of ene reductases, we tested
four different enzymes. The employed reductases were the
morphinone reductase from Pseudomonas putida M10 (MR),[36]
the NAD(P)H-dependent 2-cyclohexen-1-one reductase from
Zymomonas mobilis (NCR),[37] and the two old yellow enzymes
OYE1 and OYE3 from Saccharomyces pastorianus.[38] These four
proteins were screened with the four cofactors 1–4 and the
substrates 2-methyl-2-pentenal (5), 2-methyl-cinnamaldehyde
(6), and the isomers (E)-citral (7) and (Z)-citral (8). OYE1 and
OYE3 both accepted the synthetic cofactors and the best con-
versions were achieved with BNAH (Tables S3 and S4 in the
Supporting Information). OYE1 and OYE3 were employed in
a threefold excess relative to MR and NCR. The conversions of
OYE1 and OYE3 did not match the product formation of NCR.
This proves the literature-known excellent activity of NCR.[39]
Figure 1. The set of cofactors used in this work. The trivial names are acro-
nyms: NADH stands for nicotinamide adenine dinucleotide, PNAH for 1-
phenyl-1,4-dihydro-nicotinamide, BNAH for 1-benzyl-1,4-dihydro-nicotina-
mide, and HPNAH for 1-(4-hydroxyphenyl)-1,4-dihydro-nicotinamide.
Cyclic voltammetry
The synthesized cofactors were studied by cyclic voltammetry
to determine their electrochemical potentials and compare
them to NADH. All measured compounds exhibited an irrever-
sible oxidation peak at different potentials (Figure 2). As ex-
pected from the Hammett constants[29,30] for the inductive and
MR displayed
a preference for NADH for all substrates
(Table S2 in the Supporting Information). NCR turned out to be
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Table 1. Conversions [%] with different combinations of substrates and cofactors catalyzed by NCR.[a]
Substrates
NADH
PNAH
BNAH
HPNAH
5
68.6Æ1.2
53.7Æ5.0
54.2Æ2.6
82.0Æ3.5
6
7
8
68.7Æ1.1
33.0Æ0.5
46.8Æ9.5
65.2Æ4.8
74.0Æ1.0
62.3Æ7.8
66.1Æ2.7
68.0Æ1.6
51.3Æ6.3
95.6Æ3.1
91.6Æ6.9
88.9Æ9.3
[a] Reaction conditions: the substrate stock solution (10 mL, 100 mm in DMSO, final concentration=2 mm) and cofactor stock solution (10 mL, 125 mm in
DMSO, final concentration=2.5 mm) were added to the enzyme solution (480 mL, 50 mgmLÀ1, citrate buffer at pH 7.5, 12 mm). The mixture was stirred for
20 min at 308C.
the most active enzyme in the tested set, converting all sub-
strates >33% with the four cofactors. The best cofactor was
HPNAH for all substrates, with excellent conversions of >82%
after 20 min (Table 1). Previous work on NCR with the natural
cofactor NADH already showed a preference for the smaller
substrates 5 and 6 (conversions >68%) and lower conversions
(<47%) for the citral isomers 7 and 8.[39] Interestingly, the syn-
thetic cofactors did not display significantly lower activity with
any of the substrates.
Kinetic experiments
Because of the significant difference in the oxidation potential
of NADH and HPNAH in combination with the distinguished
reactivities with NCR and (Z)-citral 8, we performed additional
kinetic studies. As the reaction proceeds through a ping-pong-
Figure 3. Turnover frequency (TF) per minute with varied substrate and co-
factor concentrations. The blue curves refer to the systems comprising
NADH and the green lines refer to the systems comprising HPNAH. Solid
bi-bi mechanism, the cofactor as well as (Z)-citral 8 had to be
considered as substrates. Therefore, one component—either
the cofactor or the substrate—was varied in the range 0.1–
10 mm while the other was kept constant at 1 mm. As HPNAH
does not exhibit a distinguishable wavelength in either its re-
duced or oxidized form, stopped-flow monitoring was not per-
formed.
lines represent (Z)-citral 8 variation and the dashed lines represent the cofac-
tor variation
pared with that for NADH (Table 2). The reduction using a con-
stant concentration of NADH suffers a strong inhibition by (Z)-
citral 8. The Michaelis constant, Km, and the inhibition constant,
Ki, for NADH were calculated to be 0.061 mm and 0.499 mm,
respectively. The variation of NADH showed the expected be-
havior of an increased reaction rate at higher concentrations
although the enzyme showed a low affinity towards NADH
(Km =3.802 mm). With the utilization of HPNAH, the situation is
inverted. The variation of the HPNAH concentration showed
cofactor inhibition with a Ki value of 1.888 mm whereas a high
(Z)-citral 8 concentration marginally inhibited the reaction rate
with a Ki value of 14.897 mm. Additionally, the vmax value of
NADH in both systems is much lower than vmax with HPNAH.
These results indicate some guidelines for an applied reaction
setup. The NCR–NADH system demands high cofactor concen-
trations as well as low (Z)-citral 8 concentrations. Both charac-
teristics are unfavorable with respect to the needs of an effi-
cient synthesis. As cofactors are usually combined with a recy-
cling system, the apparent cofactor concentration is typically
The samples were hence extracted after 5, 10, 15, 20, and
30 min, respectively. The amount of product was thereafter de-
termined by GC-FID. The advantage of this approach in com-
parison to spectrometric determination of cofactor depletion is
the facile reaction setup. As NADH can be oxidized by oxygen,
the spectrometric setup requires a protecting gas. This is not
necessary in our approach owing to the direct detection of the
formed product rather than the indirect measurement of the
absorption decrease of the cofactor. The kinetic data revealed
that the reduction is dependent on the concentration of each
varied component (Figure 3). The variation of NADH concentra-
tion showed an expected converging increase of turnover fre-
quency at higher amounts. HPNAH acted substantially different
to NADH and showed inhibition at concentrations larger than
0.4 mm. Owing to its low solubility in water, the HPNAH con-
centration was only varied between 0.1 and 5 mm. Impressive-
ly, a threefold increase in vmax was measured for HPNAH com-
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Experimental Section
Table 2. Kinetic parameters of the reduction reaction of (Z)-citral 8 cata-
lyzed by the enzyme NCR.
Chemicals and analytics
NADH
HPNAH
Solvents and buffer components were obtained from Sigma–Al-
drich (Schnelldorf, Germany). Non-commercially available chemicals
such as the synthetic cofactors were synthesized as described
below. Analytics were carried out on a Shimadzu GC-2010-system
(Kyo¯to, Japan) with a flame ionization detector (FID) or a mass
spectrometer (MS). Helium (MS) and hydrogen (FID) were used as
carrier gases with a flow of 30 cmsÀ1 and an injection temperature
of 2508C. The injection volume was 1 mL with a split of 1:5. Quan-
tification was achieved by a calibration curve and the internal stan-
dard 1-octanol.
NADH
Z-citral 8
const.[b]
HPNAH
const.[a]
Z-citral 8
const.[b]
const.[a]
Km [mM][c]
vmax [mmolminÀ1
Ki [mM][e]
0.067
0.305
0.499
3.802
0.454
–
0.187
1.012
14.897
0.059
3.124
1.888
[d]
]
[a] Reaction conditions: the substrate stock solution (5 mL, 40 mm in
DMSO, final concentration=1 mm) and cofactor stock solution (10 mL, in
DMSO, final concentrations=0.1/0.5/1/5/10 mm) were added to the
enzyme solution (380 mL, 2 mgmLÀ1, citrate buffer at pH 7.5, 12 mm). The
mixtures were stirred for 5/10/15/20/30 min at 308C. [b] Reaction condi-
tions: the cofactor stock solution (5 mL, 40 mm in DMSO, final concentra-
tion=1 mm) and substrate stock solution (10 mL, in DMSO, final concen-
trations=0.1/0.5/1/5/10 mm) were added to the enzyme solution (380 mL,
2 mgmLÀ1, citrate buffer at pH 7.5, 12 mm). The mixtures were stirred for
5/10/15/20/30 min at 308C. [c] Km =the Michaelis constant. [d] vmax =max-
imal velocity. [e] Ki =substrate inhibition constant.
Cofactor synthesis
BNAH (3) was synthesized as previously described.[24]
1H NMR (250 MHz, CDCl3): d=3.18 (s, 2H), 4.31 (d, 2H), 4.75 (m,
2H), 5.33 (s, 2H), 5.75 (d, 1H), 7.32 ppm (m, 5H).
The syntheses of PNAH (2) and HPNAH (4) were carried out in
three steps. The first step was the preparation of Zincke’s salt (3-
carbamoyl-1-(2,4-dinitrophenyl)pyridiniumchloride). Therefore, nico-
tinamide (7.32 g, 59.9 mmol) and 2,4-dinitrochlorobenzene
(13.35 g, 65.91 mmol) were stirred for 30 min at 1008C without
a solvent. After cooling to room temperature, the resulting orange
glassy solid was dissolved in methanol (80 mL). Then, methyl tert-
butyl ether (MTBE) (50 mL) was added, the mixture was shaken,
and the upper layer was discarded. This step was repeated twice.
The precipitated solid was filtered, dissolved in methanol (20 mL),
and ethyl acetate (100 mL) was added. The solvent was discarded
and the residual solid was dried under vacuum and stored under
low, which would result in low activity of the enzyme. In the
NCR–HPNAH system, however, high substrate concentrations
(10 mm) did not lead to a significant inhibition effect and
hence allow increased substrate titres. The tolerance of high
(Z)-citral 8 concentrations in addition to the low Km of HPNAH
renders the system significantly more efficient than the natural
one.
An explanation for the observed increase in reaction speed
could be the ability of HPNAH to reduce the flavin faster than
NADH as a result of its geometry and potential. Massey et al.
identified a 20 times slower flavin reduction in OYE1 than the
subsequent reduction of cyclohexanone, which they used as
the substrate in their study.[25] In our system, both molecules—
the cofactor and the substrate—use the same active site and
are competing for binding. The combination of low steric hin-
drance and a low oxidation potential seems to allow HPNAH
to perform an accelerated reductive half reaction (k1).
1
nitrogen. H NMR (250 MHz, D2O): d=8.27 (d, 1H), 8.49 (t, 1H), 8.96
(d, 1H), 9.34 (m, 3H), 9.69 ppm (s, 1H).
The second and the third steps were carried out subsequently
without intermediate purification. 3-Carbamoyl-1-(2,4-dinitro-phe-
nyl)pyridiniumchloride (1 g, 3.08 mmol) was dissolved in methanol
(150 mL). Then, anilin (PNAH) or 4-amino-phenol (HPNAH;
3.08 mmol) were added. The obtained deep-red solution was
heated at 508C until it turned yellow. Then, the solvent was evapo-
rated and the solid was dissolved in H2O. The obtained solution
was extracted three times with MTBE (50 mL). Then, NaHCO3 (0.5 g)
was added and a nitrogen atmosphere was applied. Over the
course of 1 h, Na2S2O4 (2.3 g, 10.7 mmol) was added in portions. In
the case of HPNAH, the aqueous layer was laminated with MTBE to
ensure continuous product extraction. After the complete addition
of Na2S2O4, the solution was extracted three times with MTBE
(50 mL). The solvent was evaporated and the obtained solid was
stored under nitrogen.
Conclusions
The variation of the nicotinamide substituents led to a signifi-
cantly altered behavior. The screening of the four enzymes
NCR, MR, OYE1, and OYE3 revealed the highly active cofactor-
enzyme pair NCR–HPNAH. Cyclic voltammetric measurements
confirmed the lowered oxidation potential of HPNAH, repre-
senting a higher ability for hydride donation. This provides
a possible explanation for the increased activity. The per-
formed kinetic experiments further show that the NCR–HPNAH
system is beneficial for the synthesis of the product citronellal.
The ease of preparation and the far-reaching effects of the aro-
matic substituted nicotinamide cofactors provide an addition
to the biocatalytic toolbox. This can be perceived as a chemical
approach to synthetic biology. We term this chemical strategy
“ChemBricks” in analogy to BioBricks. The use of chemical
modification for biological challenges is still a niche area. This
niche has high potential to be applied to cofactors and co-sub-
strates such as flavins, hemes, thiamines, a-ketoglutarates, and
pyrroloquinoline quinones (PQQ).
PNAH: 1H NMR (500 MHz, DMSO): d=3.21 (d, 2H), 4.97 (m, 1H),
5.52 (s, 2H), 6.31 (m, 1H), 7.10 (m, 3H), 7.35 (m, 2H), 7.54 ppm (t,
1H).
1
HPNAH: H NMR (500 MHz, DMSO): d=3.01 (d, 2H), 4.83 (m, 1H),
6.28 (m, 1H), 6.85 (m, 5H), 7.14 (m, 2H), 9.35 ppm (s, 2H).
Cyclic voltammetry
Cyclic voltammetry was carried out in 0.1m Bu4NClO4 solutions by
using a three-electrode configuration (glassy carbon working elec-
trode, Pt counter electrode, Ag/AgCl reference) and a PAR 273 po-
tentiostat and function generator. Argon was used as the inert gas
und DMSO was used as the solvent. The ferrocene/ferrocenium
(Fc/Fc+) couple served as the internal reference.
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Enzyme preparation
For the protein expression, E. coli BL21(DE3) was transformed with
the corresponding vectors (NCR, MR, OYE1, and OYE3). Then,
a single colony was transferred to 5 mL of an overnight culture.
Terrific broth (TB) medium (400 mL) was inoculated with overnight
culture (4 mL) and incubated until an OD600 of 0.6 was reached.
Then, isopropylthiogalactopyranoside (IPTG, 0.1 mm) was added
for induction. Cells were harvested after incubation overnight at
308C and 180 rpm and resuspended in 50 mm potassium phos-
phate buffer at pH 7.5. Cells were disrupted by sonication on ice
(4”1 min, 1 min intervals). The cell debris was removed by centri-
fugation (37000 g, 45 min, 48C) and the supernatants were recov-
ered. Purification was carried out with 1 mL His GraviTrap TALON
columns and 150 mm imidazole solution as eluent. One dialysis
against tris(hydroxymethyl)aminomethane (TRIS) buffer (50 mm,
pH 7.5) for 1 h and overnight at 48C ensured the absence of imida-
zole in the purified enzyme solutions.
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Substrate screening
A stock solution of the substrate (10 mL, 100 mm in DMSO, end
concentration=2 mm) and cofactor stock solution (10 mL, 125 mm
in DMSO, end concentration=2.5 mm) were added to the enzyme
solution (480 mL, NCR/MR: 50 mgmLÀ1 and OYE1/OYE3:
150 mgmLÀ1 in citrate buffer at pH 7.5, 12 mm). The mixture was
stirred for 20 min at 308C und 180 rpm. Then, the samples were
extracted with MTBE (500 mL) and analyzed by GC-FID.
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Kinetic experiments
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Acknowledgments
The authors want to thank Prof. Jürgen Pleiss, Dr. Bettina Nestl,
and Nico Kress for their support and stimulating discussions. We
gratefully acknowledge financial support by the IMI Joint Under-
taking between the European Union and pharmaceutical industry
association EFPIA under grant agreement no. 115360-2.
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Keywords: asymmetric reduction · biocatalysis · cofactors ·
ene reductase · enzyme kinetic
Received: November 9, 2015
Published online on January 14, 2016
ChemCatChem 2016, 8, 911 – 915
www.chemcatchem.org
915
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