Donor-Acceptor Distance Sampling Enhances the Performance of "better than Nature" Nicotinamide Coenzyme Biomimetics

Article abstract of DOI:10.1021/jacs.6b05625

Understanding the mechanisms of enzymatic hydride transfer with nicotinamide coenzyme biomimetics (NCBs) is critical to enhancing the performance of nicotinamide coenzyme-dependent biocatalysts. Here the temperature dependence of kinetic isotope effects (

Full text of DOI:10.1021/jacs.6b05625

Communication
pubs.acs.org/JACS
Donor−Acceptor Distance Sampling Enhances the Performance of
“Better than Nature” Nicotinamide Coenzyme Biomimetics
Alexander Geddes,† Caroline E. Paul,‡ Sam Hay,† Frank Hollmann,‡ and Nigel S. Scrutton*,†
†BBSRC/EPSRC Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biology
and School of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom
‡Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628BL Delft, The Netherlands
S
* Supporting Information
synthetic NCBs, and coenzyme analogue recycling to
ABSTRACT: Understanding the mechanisms of enzy-
matic hydride transfer with nicotinamide coenzyme
biomimetics (NCBs) is critical to enhancing the perform-
ance of nicotinamide coenzyme-dependent biocatalysts.
Here the temperature dependence of kinetic isotope
effects (KIEs) for hydride transfer between “better than
nature” NCBs and several ene reductase biocatalysts is
used to indicate transfer by quantum mechanical
tunneling. A strong correlation between rate constants
and temperature dependence of the KIE (ΔΔH⧧) for H/D
transfer implies that faster reactions with NCBs are
associated with enhanced donor−acceptor distance
sampling. Our analysis provides the first mechanistic
insight into how NCBs can outperform their natural
counterparts and emphasizes the need to optimize donor−
acceptor distance sampling to obtain high catalytic
performance from H-transfer enzymes.
demonstrate the overall effectiveness of NCBs in ER-catalyzed
biotransformations.14 Some NCBs outperform natural coen-
zymes in the reduction of selected ERs.14 The origin(s) of this
enhanced catalytic performance is unknown. This knowledge is
important for understanding of the physical basis of catalysis
and also for the development of related biomimetics with high
catalytic potential for other oxidoreductases.16−19
The ERs have been the subject of intensive study from the
viewpoint of H-transfer,20,21 aided in part by the availability of
high-resolution crystal structures of several ERs and substrate/
ligand complexes and the accessibility of the reaction cycle that
comprises two half-reactions (Figure 1).14,22−28 The kinetics of
the reaction cycle have been derived using a number of ER−
substrate combinations and, in selected cases, extended to
obtain free energy profiles. Hydride transfer from natural
nicotinamide coenzymes (NADH and/or NADPH) to the
enzyme-bound flavin (flavin mononucleotide, FMN) involves
significant quantum mechanical tunneling (QMT).27 Temper-
ature-dependent kinetic isotope effects (KIEs) on a range of
enzymatic H-transfer reactions has led to the promoting
vibrations hypothesis, where enzyme/substrate dynamics
(conformational/distance sampling) are coupled to the H-
transfer coordinate29−32a model that has been developed
extensively in relation to ER-catalyzed H-transfers.27,33,34
Here we set out to ascertain the origin of the enhanced
performance of selected NCBs/ER combinations by studying
the isotope dependence of reaction rate as a function of
temperature.20,21,28,35 The temperature dependence of KIEs is a
key descriptor for distinguishing between semiclassical and QM
mechanisms of transfer and for uncovering the relative and
inferred importance of distance sampling/conformational
coupling to the reaction coordinate.29,32
The temperature dependence of hydride transfer during the
reductive half-reactions of the three ERs [PETNR, XenA, and
thermophilic OYE (TOYE)] was measured with saturating
concentrations of four coenzymes and NCBs [NADH,
NADPH, 1-benzyl-1,4-dihydronicotinamide (mNH2), and 1-
butyl-1,4-dihydronicotinamide (mBu)] by stopped-flow spec-
troscopy that monitors the loss of oxidized FMN absorbance at
464 nm. Observed rate constants are given in Tables S1−S7,
and Eyring plots of these data are shown in Figure 2 (and in
expanded format in Figures S1−S3). The observed rate
he search for synthetic biomimetics that can replace
Tnatural nicotinamide coenzymes has been driven by the
instability and expense of NAD(P)H. This has prevented
widespread use of natural coenzymes, for example, in
biocatalytic manufacture of fine and specialty chemicals. The
use of in situ regeneration systems to replenish natural
coenzymes during catalytic turnover,1−6 or through the use of
hydrogen borrowing biocatalytic cascades,7−11 is one solution;
an alternative is to develop stable synthetic nicotinamide
coenzyme biomimetics (NCBs) that can be regenerated and
that have the potential to operate generally with biological
oxidoreductases that normally function with NAD(P)H.12 The
development of such NCBs would be a game-changer in the
green manufacture of chemicals. NCBs that satisfy these
requirements are beginning to emerge. They offer hope for
chemicals manufacture using oxidoreductases that catalyze a
wide range of chemical transformations.13
NCBs support biocatalysis with many ene reductases
(ERs),12,14 which belong to the Old Yellow Enzyme (OYE)
family (EC 1.3.1.31). These are broad specificity oxidoreduc-
tases that catalyze the asymmetric reduction of activated CC
bonds.15 Their broad specificity and ability to introduce new
stereogenic centers makes them attractive targets for industrial
biocatalysis. Cognizant of these properties, we have reported
crystal structures of selected ER-NCB complexes, a compre-
hensive analysis of reactions catalyzed by 12 ERs with 5
Received: June 1, 2016
© XXXX American Chemical Society
A
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Communication
constants vary by 200-fold at 25 °C, from 2 s−1
(PETNR:NADH) to 434 s−1 (TOYE:mBu). In all 3 enzymes,
NADH is the slowest substrate, and mBu is the fastest. The
apparent activation enthalpy (ΔH⧧) varies by a factor of 2.5-
fold from 22.8 1.2 kJ mol−1 (TOYE:mBu) to 58.5 1.8 kJ
mol−1 (XenA:NADH) (Figure 3; Tables S7−S10). Some
Figure 3. Relationship between apparent activation enthalpy and
activation entropy determined from the Eyring plots in Figure 2. Black,
red, and blue points correspond to those reactions with PETNR,
TOYE, and XenA, respectively, using NADH (squares), NADPH
(circles), mNH2 (up-triangles), and mBu (down-triangles). Filled and
hollow points correspond to those reactions with protiated and
deuterated coenzymes, respectively.
Figure 1. Catalytic cycle of PETNR and structures of the natural
coenzymes and synthetic biomimetics. A) Reductive half-reaction,
involving hydride transfer from the nicotinamide C4 pro-R position of
NAD(P)H, or synthetic mimics, to the FMN N5 resulting in bleached
flavin absorbance at 464 nm. The cycle culminates with hydride
transfer from the dihydroFMN N5 and proton transfer from solvent to
the oxidative substrate. B) The structure of natural coenzymes,
NADPH and NADH, and the synthetic coenzyme mimics, 1-benzyl-
1,4-dihydronicotinamide (mNH2) and 1-butyl-1,4-dihydronicotina-
mide (mBu).
enthalpy−entropy compensation is apparent, and the apparent
activation entropy (ΔS⧧) varies from −124 2 kJ mol−1 K−1
(TOYE:mNH2) to −42 6 kJ mol−1 K−1 (XenA:NADH).
In the TOYE and XenA reactions, there is some correlation
between log kobs and ΔH⧧ for hydride (Figure 4) and deuteride
(Figure S7) transfer, consistent with both transition state
theory (TST) and models of enzymatic non-adiabatic H-
transfer (i.e., by QMT). The PETNR reactions do not show
Figure 2. Eyring plots (top panels) and temperature dependence of the KIEs (bottom panels) on the reactions of PETNR (A), TOYE (B), and
XenA (C) with natural coenzymes and synthetic coenzyme mimics NADH (green), NADPH (orange), mNH2 (red), and mBu (blue). Data are
fitted to the Eyring equation, with fitted parameters shown in Figure 3 and given in Tables S7−S10. Only protiated data are shown in the Eyring
plots here; all data are shown in Figures S1−S3.
B
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reductant (XenA:mBu, TOYE:mBu, PETNR:mBu, Xe-
nA:mNH2, and PETNR:mNH2) have reactions with ΔΔH⧧ >
10 kJ mol−1. This is good evidence for H transfer by significant
QMT, as such large values of ΔΔH⧧ are inconsistent with
either TST or Bell-type models of H-transfer where QMT
occurs near the transition state.37,38
In all three enzymes, there is some correlation between
observed rate constant and ΔΔH⧧ (Figure 4), with the faster
reactions typically having more temperature-dependent KIEs.
This suggests that whatever gives rise to the temperature
dependence of the KIEse.g., conformation/distance sampling
within the promoting vibrations hypothesismay also enhance
the rate of H-transfer/FMN reduction in the three ERs
investigated here. As the most temperature-dependent KIEs are
observed on reactions involving NCBs, an alternative
explanation is that the differences in kinetics arise through
chemical differences between the coenzymes and NCBs.
Density functional theory models of NAD(P)H and the two
NCBs show that, while the C4−H stretching frequencies and
gas-phase bond dissociation energies (Figure S9) are very
similar between NCBs and natural coenzymes, the methyl-
benzene and butyl (“tail”) moieties of the NCBs have greater
electron-withdrawing properties than the (2′-phospho)-
adenosine diphosphate ribose moieties of NAD(P)H (Table
S12). This is expected to facilitate oxidation of the NCBs due
to stabilization of positive charge buildup in the oxidized
species. The redox potentials of NAD(P)H, mNH2, and the
mBu analogue 1-propyl-1,4-dihydronicotinamide have been
reported.13,39 These are in qualitative agreement with our DFT
calculations (Table S12). The likely order of reduction
potentials are NADPH = NADH > mNH2 > mBu, consistent
with hydride self-exchange reactions in acetonitrile with the
reactivity order mBu > mNH2 > NAD(P)H. The reduced
reduction potentials/increased driving force of NCBs relative to
natural coenzymes may explain some of the observed rate
enhancement of the ER-NCB reactions. However, the
reduction potentials of NADH and NADPH are not
significantly different, yet the reactions of all three ERs with
NADPH are 10−20-fold faster than with NADH. Further, in
both PETNR and XenA, NADPH reacts as fast as or faster than
mNH2, despite the likely larger driving force of the mNH2
reaction.40 Modest changes to the reaction driving force are
also not expected to alter the magnitude of the KIE,31,41 so the
kinetic differences observed between natural and biomimetic
coenzymes do not appear to solely arise through differing
reaction driving forces.
Figure 4. Relationship between ΔH⧧H (upper panel; equivalent plot
for ΔH⧧D given in Figure S7) and ΔΔH⧧ (ΔΔH⧧ = ΔH⧧D − ΔH⧧H
;
lower panel) and the observed rate constant for each hydride transfer
reaction at 25 °C. Black, red, and blue points correspond to those
reactions with PETNR, TOYE, and XenA, respectively, using NADH
(squares), NADPH (circles), mNH2 (up-triangles), and mBu (down-
triangles).
this correlation, suggesting that the origin(s) of the reduction in
activation enthalpy on the TOYE and XenA reactions must
involve the enzyme and that the reactions do not solely
originate from differing properties of the coenzymes/NCBs. In
all cases, there is less correlation between log kobs and ΔS⧧
(Figure S6), suggesting that enthalpy effects dominate the rate
of reaction.
The primary KIEs on these 12 reactions were measured using
(R)-[4-2H]-NADH, (R)-[4-2H]-NADPH, [4-2H2]-mNH2, and
[4-2H2]-mBu (Figure 2). Dideuterated NCBs were synthesized
by sequential dithionite reduction, chloranil oxidation, and re-
reduction with dithionite in D2O as described (Supporting
Information). NMR indicated the NCBs were 98% dideu-
terated at C4. There is relatively little variation in the
magnitude of the observed KIE at 25 °C (Figure 2). All KIEs
fall within the range of 7−9, except those measured with XenA
and the natural coenzymes: KIE = 4.74 0.13 and 5.09 0.48
with NADH with NADPH, respectively. The α-secondary
deuterium KIEs have been previously measured on several OYE
ERs including PETNR. The observed KIEs on the PETNR
reactions with (S)-[4-2H]-NADH and (S)-[4-2H]-NADPH are
1.16−1.20 and are not significantly temperature dependent.36
The α-2° KIEs on the reactions of the ERs with mNH2 or mBu
are unlikely to be larger than 1.20. The observed KIEs
measured with dideuterated mNH2 and mBu are thus unlikely
to exceed the primary KIE by more than 20%.
ER active sites are evolutionarily constrained by the necessity
to bind both oxidative and reductive substrates within the same
active site region, thus the evolution of improved NAD(P)H
binding may have been constrained to the binding of the
coenzyme “tail” on the periphery of the active site, beyond the
reach of oxidative substrates.22−26 While the tail allows
coenzyme capture, the Km and KS values are relatively large
(cf. other flavoproteins where the coenzyme adenosyl ribose
moiety is a key factor in binding).42 We hypothesize that by
removing/reducing the size of the “tail”, NCBs can bind and
react in a manner more similar to the oxidative substrates of
ERs. These differences must be subtle as X-ray crystal
structures of NCB-and coenzyme-bound ERs show the
nicotinamide moieties to be essentially superimposed (Figures
S10 and S11).
The temperature dependence of the KIEs varies widely from
0−18 kJ mol−1 (PETNR:NADH and XenA:mBu, respectively;
Figures 2,4, S4, and S5). Five combinations of enzyme/
NCBs typically have lower activation enthalpies compared to
their natural counterparts (Figure 3). However, this is at the
C
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b05625.
Experimental details and characterization data, including
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AUTHOR INFORMATION
■
Corresponding Author
*nigel.scrutton@manchester.ac.uk
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Supported by the UK Biotechnology and Biological Sciences
Research Council (BBSRC; BB/M017702/1) and Bruker UK
Ltd. (Ph.D. studentship to A.G.). N.S.S. is an Engineering and
Physical Sciences Research Council (EPSRC) Established
Career Fellow (EP/J020192/1).
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