Biocatalyst-artificial metalloenzyme cascade based on alcohol dehydrogenase

Article abstract of DOI:10.1039/C8SC02371A

Chemo-enzymatic cascades of enzymes with transition metal catalysts can offer efficient synthetic strategies, but their development is challenging due to the incompatibility between proteins and transition metal complexes. Rhodium catalysts can be combined with alcohol dehydrogenases to regenerate nicotinamide cofactors using formate as the hydride donor. However, their use is limited, due to binding of the metals to residues on the enzyme surface, leading to mutual enzyme and catalyst inactivation. In this work, we replaced the zinc from Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) with Rh(iii) catalysts possessing nitrogen donor ligands, by covalent conjugation to the active site cysteine, to create artificial metalloenzymes for NADP⁺ reduction. TbADH was used as protein scaffold for both alcohol synthesis and the recycling of the cofactor, by combination of the chemically modified species with the non-modified recombinant enzyme. Stability studies revealed that the incorporation of the catalysts into the TbADH pocket provided a shielding environment for the metal catalyst, resulting in increased stability of both the recycling catalyst and the ADH. The reduction of a representative ketone using this novel alcohol dehydrogenase-artificial formate dehydrogenase cascade yielded better conversions than in the presence of free metal catalyst.

Full text of DOI:10.1039/C8SC02371A

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Biocatalyst – artificial metalloenzyme cascade based on alcohol
dehydrogenase
Simone Morra a,b and Anca Pordea*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Chemo-enzymatic cascades of enzymes with transition metal catalysts can offer efficient synthetic strategies, but their
development is challenging due to the incompatibility between proteins and transition metal complexes. Rhodium
catalysts can be combined with alcohol dehydrogenases, to regenerate nicotinamide cofactors using formate as the
hydride donor. However, their use is limited, due to binding of the metals to residues on the enzyme surface, leading to
mutual enzyme and catalyst inactivation. In this work, we replaced the zinc from Thermoanaerobacter brockii alcohol
dehydrogenase (TbADH) with Rh(III) catalysts possessing nitrogen donor ligands, by covalent conjugation to the active site
cysteine, to create artificial metalloenzymes for NADP+ reduction. TbADH was used as protein scaffold for both alcohol
synthesis and the recycling of the cofactor, by combination of the chemically modified species with the non-modified
recombinant enzyme. Stability studies revealed that the incorporation of the catalysts into the TbADH pocket provided a
shielding environment for the metal catalyst, resulting in increased stability of both the recycling catalyst and the ADH. The
reduction of a representative ketone using this novel alcohol dehydrogenase – artificial formate dehydrogenase cascade
www.rsc.org/
yielded
better
conversions
than
in
the
presence
of
free
metal
catalyst.
are chemical catalysts based on Rh(III), Ir(III) and to a lesser
extent Ru(II), which use formate as a reducing agent.7-12
Although less active than formate dehydrogenase (turnover
frequency ~250 times lower for NADH formation for the state-
of-the-art Rh complex),3,13 they are compatible with high
temperatures and solvents, and avoid the complexity
associated with a second enzyme. Additionally, organometallic
complexes have been shown to recycle the simpler and less
expensive synthetic NAD(P)H mimics.14,15 Sadler and co-
workers also reported the formate-driven reduction of NAD+ to
NADH catalysed by Ru(II) arene complexes in living cells, albeit
with reduced activity compared to the complexes in buffer.16
The main limitation of these synthetic regeneration catalysts is
the incompatibility of the metal complexes with coordinating
functionalities present in the mixture, such as accessible amine
or thiol groups present in enzymes.17-19 Restricting the access
of the metal species to the nucleophilic residues on the
enzyme surface is required to solve this problem. Attempts to
protect the catalysts by separately immobilising the enzyme
and the metal complex have been shown to increase process
stability, and offered opportunities for catalyst recycling.4,6 On
the other hand, immobilisation of the catalysts resulted in
decreased catalytic activity due to diffusion limitations.20
1 Introduction
Alcohol dehydrogenases (ADHs) are valuable biocatalytic tools
for the conversion of ketones into chiral alcohols, providing a
sustainable and efficient alternative to chiral catalysts based
on transition metals.1 The main limitation to their practical
application is the requirement of nicotinamide adenine
dinucleotide cofactors (NADH or NADPH) as electron donors.
The complexity of these structures, associated with high cost
and reduced stability, precludes their use as stoichiometric
reducing agents. Therefore, the continuous in situ
regeneration of the cofactor is necessary for economically
acceptable transformations with isolated enzymes. Current
state of the art for NAD(P)H recycling is the use of a second
catalyst to regenerate the cofactor at the expense of another
reducing agent.2 Enzymatic recycling systems have been
developed, inspired by the mechanisms of cofactor balance in
living cells, but they are not universally applicable and may
suffer from incompatibility between the two enzyme systems
and complexity of product purification.3
Both chemical and electrochemical delivery of reducing
potential have been investigated, as simpler and more robust
alternatives to enzymatic methods.2-6 A very promising option
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Figure 1. Reduction of a model ketone using a native – artificial enzyme cascade with the same alcohol dehydrogenase scaffold. L – L represents
a bidentate ligand. The protein is represented in green, and the rhodium complex is represented in red.
As an alternative to the immobilisation on solid support, can lead to exquisite control over the activation pathway, and
artificial metalloenzymes are constructed by encapsulation of thus to promiscuous reactivities, as demonstrated by Hyster
metal-based catalysts inside protein scaffolds, thus and co-workers who used an NADPH-dependent reductase to
overcoming mass transfer limitations by keeping the catalytic catalyse radical formation and hydrogen atom transfer.34 Zinc-
moiety and the reagents in homogeneous phase. They have dependent alcohol dehydrogenases are well-suited to build
been explored as a synergistic method in catalyst design, by artificial enzymes for NAD(P)+ reduction: they contain a metal-
coupling the selectivity of enzymes with versatile, human- binding site, a hydrophobic substrate-binding pocket able to
devised, catalytic functionality, to afford synthetic capabilities accommodate organic ligands, as well as the NAD(P)+ cofactor-
greater than the sum of the parts.21 Artificial metalloenzymes binding pocket.35 In this study, we will present the
have been shown to facilitate a range of synthetic reaction development of hybrid catalysts for NADPH regeneration, by
chemistries, from reductions of carbon-carbon and carbon- using a zinc-dependent ADH as host for metal complexes with
heteroatom bonds, to oxygen insertion and carbon-carbon demonstrated ability to reduce NAD(P)+. In the native ADH
bond formation.21-27
reaction, the reduction of NAD(P)+ takes place by hydride
The reduction of NAD(P)+ has also been achieved with metal transfer from an alcohol hydride donor. We reasoned that the
catalysts incorporated into proteins, either by covalent linking replacement of the alcohol by a metal hydride donor will
to papain,28 or by supramolecular insertion using the well- provide the advantage of irreversible hydride transfer from
established biotin-streptavidin system.29 In this context, Ward formate to the cofactor. We hypothesise that the use of ADH
and co-workers reported that the protein host also provided a as the protein scaffold for both alcohol synthesis (natural ADH)
shielding environment for the metal catalyst against inhibitory and the recycling of the cofactor (chemically modified ADH)
species present in solution. This concept of “molecular will result in an increased robustness of a system where both
compartmentalisation” has been demonstrated by an reactions take place in the same mixture (Figure 1). To test this
increased activity of the protein-bound compared to the free hypothesis, the potential of the protein to prevent metal
metal complex, when used in situ with various enzymes inactivation will be studied and the new artificial
dependent on NAD(P)H or on NAD(P)H mimics.30-32 Whilst a metalloenzymes will be compared with non-enzymatic
range of cofactor-dependent biocatalysts have been employed cofactor regeneration catalysts in a synthetic cascade.
in conjunction with artificial metalloenzymes, such as ene
reductases, glucose dehydrogenase and 2-hydroxybiphenyl
2 Results and discussion
monooxygenase,
a
cascade reaction with alcohol
dehydrogenases remains to be developed. This is most likely
due to the ketone reduction activity of the most successful
biotinylated Cp*Ir aminosulfonamide complexes reported so
far, which would interfere with the ADH activity.33
2.1 Design and preparation of alcohol dehydrogenase mutants
ADH from the thermophilic species Thermoanaerobacter
brockii (TbADH) was selected for bioconjugation to a metal
complex catalyst. TbADH is a Zn-dependent bacterial ADH with
a homotetrameric structure, in which the catalytic Zn is bound
by residues C37, H59 and D150.36 Its distinguishing feature
compared to other medium-chain Zn-dependent ADHs is the
In most cases, the design of artificial metalloenzymes relies on
the availability of a bioconjugation site within the protein
scaffold, whilst substrate-binding features are less important
in the design. Yet, substrate binding in an enzyme active site
2 | J. Name., 2012, 00, 1-3
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Figure 2. (A) Crystal structure of TbADH complexed to NADP+ (pdb 1YKF). Cysteine residues are highlighted in magenta, active site residues H59 and D150
are highlighted in green and the catalytic zinc ion is shown as a grey sphere; (B) Engineering of TbADH for the covalent binding of small molecules into the
active site. Absolute values for WT TbADH were as follows: specific activity (2-butanol oxidation coupled to NADP+ reduction, U = ꢀmol min-1): 74 ± 7 U mg-1
;
free thiols: 0.98 ± 0.003 per monomer; Zn content: 0.64 ± 0.02 atoms per monomer.
absence of a second, structural Zn-binding site. Wild-type would fit within the Zn cavity, and in proximity of the
TbADH reduces small aliphatic ketones with high nicotinamide C4 redox site.
enantioselectivity.37,38 Of particular interest is its NADP+
2.2 Bioconjugation of metal complexes to TbADH
specificity, as well as its stability at relatively high
temperatures (up to 65-86 ᵒC)39 and in the presence of organic For this study, rhodium catalysts of the type
solvents.37,40 It was reasoned that its robustness would [Cp*RhIII(N^N)(H2O)]2+ were selected, which were previously
facilitate the introduction of organic ligands within the shown to recycle NAD(P)H with formate as the hydride donor
structure. Covalent anchoring to cysteine was selected as the (N^N = chelate nitrogen donor ligands).7,9 Bipyridine and
method for the bioconjugation of metal catalysts. TbADH phenanthroline ligands containing a bromide functionality
contains four cysteine residues per monomer (Figure 2A). Two (Scheme 1), and their corresponding metal complexes
of these have already been covalently labelled with iodoacetic [Cp*Rh(Br-L1)Cl)]Cl and [Cp*Rh(Br-L2)Cl)]Cl, were prepared
acid: C203 in the NADP+ binding pocket and C37 in the Zn- according to published procedures (see ESI). Bromide was
binding site, which could be readily modified in the absence of chosen as the alkylating functionality, because this allowed the
catalytic Zn. Previous work also showed that C203 could be design of the shortest possible anchor between the protein
mutated to serine without loss in enzyme activity.41
WT TbADH was overexpressed in E. coli and purified by affinity The bioconjugation of brominated ligands to TbADH was
chromatography. The specific activity of this recombinant assessed by ESI-MS. Labelling of TbADH 5M with Br-L2
and the metal complex.
,
enzyme was comparable to the native enzyme.39 The containing the bromo acetamide anchor, occurred readily at
accessibility of the cysteine residues and their reactivity pH 7, yielding exclusively the mono-labelled protein. The
towards 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) was modification with Br-L1, bearing an alkyl bromide functionality,
assessed by Ellman’s assay, which indicated the availability of required an increased pH of 8 to achieve more than 50 % yield,
one thiol per enzyme monomer in WT TbADH, presumably and also resulted in unselective modification at a second site
C203 according to the structure and previous evidence.41 The (see ESI). Free thiols depletion after conjugation was assayed
triple mutant C203S-C283A-C295A (hereafter TbADH 3M), by the Ellman’s assay and was in good agreement with the
obtained by site-directed mutagenesis, was expressed and
purified, showing similar specific activities compared to WT
TbADH (Figure 2B), thus suggesting that removal of these thiol
groups does not significantly alter the native protein structure.
This mutant showed no free thiol content, indicating that C37
was not available for modification in the presence of zinc. Zinc
removal was achieved by further mutation of the Zn-
coordinating residues to smaller alanine side chains. As
expected and confirmed by ICP-MS analysis, double mutant
H59A-D150A contained no zinc and its reaction with the
Ellman’s reagent showed the availability of 1.6 free thiols per
monomer for covalent modification. Thus, a working TbADH
scaffold containing the five mutations H59A-D150A-C203S-
C283A-C295A (hereafter TbADH 5M) was prepared, devoid of
zinc and containing C37 as the only reactive free thiol (Figure
2B). It was hypothesized that a metal complex attached to C37
Scheme 1 Chelate nitrogen donor ligands used in this study.
residual amount of free 5M observed by ESI-MS.
When the corresponding rhodium complexes were used, the
alkylation at pH 7 was more specific than at pH 8 or 9,
presumably because of unspecific rhodium binding to
deprotonated surface residues at the higher pH. The optimum
conditions for the formation of the mono-labelled proteins as
the major compounds were obtained by incubating TbADH 5M
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Figure 3. Characterisation of covalently labelled TbADH variants, [Cp*Rh(5M-C37L1)]2+ and [Cp*Rh(5M-C37L2)]2+ by (A) ESI-TOF mass spectrometry and
(B) UV-Vis spectroscopy. UV-Vis analysis was performed in sodium phosphate buffer (100 mM, pH 7), at 10 µM concentration (determined by Bradford
assay for the proteins, and by weight for the free rhodium complexes).
with 4 equivalents of the metal complex in 100 mM Tris-HCl and the bioconjugated metal complexes, indicating a similar
buffer at pH 7, for 1 h at room temperature. The proteins were primary coordination sphere around the rhodium centre.
subsequently purified by removing the excess labelling agent To assess non-covalent binding of the catalysts outside the
on PD-10 desalting columns. Using this procedure, [Cp*Rh(5M- active site, the zinc-containing TbADH 3M mutant was treated
C37L1)]2+ was obtained as a mixture with non-labelled 5M, with the non-brominated Rh catalysts [Cp*Rh(H-L1)Cl)]Cl and
while [Cp*Rh(5M-C37L2)]2+ was obtained as a mixture with the [Cp*Rh(H-L2)Cl)]Cl. Binding of 0.5 equiv. of rhodium to the
di-labelled protein (Figure 3A). Additionally, MALDI-TOF protein was still observed by ICP-MS, although at a different
analysis of the peptides obtained after protein digestion with site from C37, as indicated by the almost completely retained
chymotrypsin confirmed the covalent labelling at C37 (see ESI). ketone reduction activity of the 3M mutant (data not shown).
Protein concentration was determined by Bradford assay. This suggested that di-labelling of the protein was due to
Labelled and unlabelled protein samples of the same rhodium coordination, rather than covalent modification of
concentrations also showed similar SDS-PAGE band intensities, residues other than cysteines. Purification by ultrafiltration,
thus excluding interference of the metal complex with the extensive dialysis or by passing through desalting columns
assay. Inductively coupled plasma mass spectrometry (ICP-MS) could not remove the Rh complex, suggesting its strong
analysis showed an average Rh : protein molar ratio of 0.5 : 1 binding to the protein. Literature has already reported the
in both cases. In subsequent experiments, unlabelled protein binding of up to 4 equivalents of [Cp*Rh(bipy)H2O)]2+ to the
concentration was determined by Bradford, whilst labelled thermostable TADH, potentially to surface histidine and
protein concentration was determined from the Rh ICP-MS cysteine residues, without influencing the catalytic activity of
analysis.
the enzyme.17 Here, we observed a similar propensity of
Characterisation of the labelled proteins by UV-Vis TbADH to unspecifically bind rhodium complexes. Given that
spectroscopy indicated the presence of the Rh complex in both formation of a single TbADH species covalently bound to one
protein samples. The UV-Vis spectrum of [Cp*Rh(5M-C37L1)]2+ rhodium complex could not be achieved, we proceeded with
showed an absorption band at 323 nm, which could not be the evaluation of the mixture characterised above.
observed in the non-labelled 5M mutant and was very similar
to the absorption of free [Cp*Rh(Br-L1)]2+. Similarly, an
2.3 Formate dehydrogenase activity of artificial metalloenzymes
increase in absorbance compared to 5M was observed for the The ability of the new artificial metalloenzymes to reduce
bioconjugated [Cp*Rh(5M-C37L2)]2+ above 300 nm, indicating NADP+ using formate as the hydride source was evaluated
the presence of the rhodium-phenanthroline complex (Figure next, by following the increase of absorbance at 340 nm, due
3B). The characteristic absorbance was very similar for the free to the formation of NADPH (Table 1). Interestingly, a lag phase
could be observed for about 20-30 seconds at the beginning of
4 | J. Name., 2012, 00, 1-3
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the reactions, during which the enzyme activity was negligible, bioconjugated Rh-bipyridine complex (Table 1, entry 5), but
after which time the reaction started (see ESI). This might be not when the phenanthroline ligand was used. This result does
due to the shielding effect of the protein matrix, slowing down not reflect the NADP+-specificity of WT TbADH (specific activity
the initial attachment of either formate or NADP+ to the is approx. 120 times higher with NADP+ than with NAD+, see
catalyst. The turnover frequency (TOF) at 50 ᵒC was calculated ESI). This is likely due to the rhodium hydride in the artificial
from the reaction rate measured during linear absorbance metalloenzyme being more exposed than the zinc-bound
increase. TOF was defined as the quantity of NADPH formed alcoholate catalytic species in the wild-type enzyme, and
per unit of time (calculated using ε340 = 6220 M-1 cm-1), and possibly rearranging the cofactor binding.
divided by the quantity of the rhodium catalyst (determined by
ICP-MS).
Figure 4. Inactivation of rhodium catalysts by TbADH. (A) Non-conjugated [Cp*Rh(H-L1)Cl]Cl; (B) Bioconjugated [Cp*Rh(5M-C37L1)]2+; (C) Non-conjugated
[Cp*Rh(H-L2)Cl]Cl; (D) Bioconjugated [Cp*Rh(5M-C37L2)] 2+. Rhodium catalysts (10 ꢀM) were incubated with 1 equiv. WT TbADH (10 ꢀM) in sodium
phosphate buffer (100 mM, pH 7) at 50 ᵒC. Catalyst activity was determined by monitoring NADPH formation at 340 nm in the presence of formate (100
mM) at 50 ᵒC. In the absence of TbADH after 1 h heating, TOFRh were as follows: 147 ± 1 h-1 for [Cp*Rh(H-L1)Cl]Cl; 28 ± 1 h-1 for [Cp*Rh(5M-C37L1)]2+; 112 ±
2 h-1 for [Cp*Rh(H-L2)Cl]Cl; 38 ± 1 h-1 for [Cp*Rh(5M-C37L2)]2+
.
Table 1. Reduction of NADP+ by Cp*Rh complexes in the presence of formate.
2.4 Compatibility between bioconjugated Rh catalysts and TbADH
The mutual inactivation between the Rh-based catalysts and
wild-type TbADH was assessed after incubation at 50 ᵒC for 1 h
and for 24 h (Figure 4). In the presence of 1 equiv. TbADH (Rh :
TbADH molar ratio = 1 : 1), the free catalysts lost most of their
activity after incubation at 50 ᵒC. This is in line with previously
published data by Hollmann and co-workers, who
demonstrated complete inactivation of up to 4 equivalents of
[Cp*Rh(Bipy)H2O)]2+ after incubation for 1 h with TADH from
Thermus ATN1 species, suggesting the presence of four Rh
binding sites within this enzyme.17 In our work, some residual
Rh activity was still observed after 1 h incubation, suggesting
that the inactivating effect on the rhodium complex was less
significant with TbADH than with TADH. However, the activity
of the complexes after incubation with TbADH was far lower
than in the absence of the enzyme.
Entry
Catalyst
Substrate
NADP+
TOFRh (h-1)
1
2
3
4
5
[Cp*Rh(Br-L1)Cl)]Cl
[Cp*Rh(Br-L2)Cl)]Cl
248
207
59
NADP+
[Cp*Rh(5M-C37L1)]2+ NADP+
[Cp*Rh(5M-C37L2)]2+ NADP+
74
[Cp*Rh(5M-C37L1)]2+ NAD+
36
+
)
Catalysis conditions: NAD
(12.5
(
P
(0.15 mM), sodium formate (500 mM), Rh catalyst
M with free catalyst) in buffer
ꢀ
M with artificial metalloenzymes; 25
ꢀ
(sodium phosphate, 100 mM, pH 7) at 50 ᵒC. The reduction rate was measured
by monitoring the absorbance at 340 nm.
The activity of the brominated free catalysts [Cp*Rh(Br-
L1)Cl)]Cl and [Cp*Rh(Br-L2)Cl)]Cl was similar to previous
literature results (entries 1-2).7,13 When covalently anchored
to TbADH, the rhodium catalysts were less active than in their
free form (entries 3-4), however their activities were
When the bioconjugated catalysts were incubated with 1
equiv. TbADH, an increased stability was observed compared
to the free complexes, with 42 % and 33 % residual activity
observed after 1 h incubation for [Cp*Rh(5M-C37L1)]2+ and
[Cp*Rh(5M-C37L2)]2+, respectively. This observation confirms
that the protein scaffold acts as a shielding environment
around the Rh catalysts, protecting these from deactivation by
comparable
with
previously
published
artificial
metalloenzymes for NAD+ reduction.28,30 Such a decrease in
activity between free and protein-embedded catalysts has
previously been observed for the NADH regeneration with
biotinylated Cp*Ir complexes incorporated into streptavidin.30
Some specificity for NADP+ vs NAD+ was observed with the
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Table 2. Reduction of 4-phenyl-2-butanone using TbADH and Rh-based NADPH
regeneration catalysts.
unspecific interactions with residues on the surface of the
enzyme. Higher Rh / TbADH ratios resulted in increased
relative activity of the complexes (see ESI).
Entry
Regeneration catalyst
Conversion (%)
The beneficial effect of bioconjugation was even more
prominent in the case of TbADH deactivation by the Rh
catalysts. When exposed to 5 equivalents of Rh catalyst for 1 h,
1
2
3
4
5
-
0
[Cp*Rh(5M-C37L1)]2+
[Cp*Rh(H-L1)Cl)]Cl
[Cp*Rh(5M-C37L2)]2+
[Cp*Rh(H-L2)Cl)]Cl
67
57
71
58
Catalysis conditions: 4-phenyl-2-butanone (5 mM), NADP+ (0.5 mM), sodium
formate (500 mM), Rh catalyst (25 ꢀM), WT TbADH (1.5 M) in buffer (sodium
ꢀ
phosphate, 100 mM, pH 7) at 50 ᵒC. Conversion and enantioselectivities were
determined after 24 h by chiral HPLC, using a CHIRALCEL OD column. Conversion
are presented as averages of triplicate runs, and standard deviations were < 1 %.
Enantioselectivities were > 99 % ee in favour of the (S)-enantiomer, in all cases.
The reduction of 4-phenyl-2-butanone by TbADH was tested at
50 ᵒC for 24 h, using 0.5
% (mol) of either free or
bioconjugated NADPH regeneration catalysts (see Figure 1).
Given the much lower turnover frequencies of Rh catalysts (~
0.02 s-1) compared to TbADH (~ 2 s-1 for 2-butanone, data not
shown), the amount of TbADH necessary to perform the
transformation should be orders of magnitude lower than the
amount of Rh. However, in line with stability studies, TbADH
Figure 5. Effect of the free and bioconjugated rhodium catalysts on 2-butanone
reduction by WT TbADH. WT TbADH (1 ꢀM) was incubated with the corresponding Rh
catalyst (5 ꢀM) in sodium phosphate buffer (100 mM, pH 7). In the absence of the Rh
catalyst, the specific activity of WT TbADH (2-butanone reduction coupled to NADPH
inactivation occurred at concentrations < 1
ꢀ
in the use of an optimised ratio of 25 M Rh and 1.5
M, which resulted
M WT
ꢀ
ꢀ
TbADH. The extent of inactivation was lower when
bioconjugated catalysts were used (see ESI). Although the
initial activity of the free Rh catalysts for NADP+ reduction was
higher (see Table 1), the bioconjugated catalysts had better
overall performance for the reduction of the ketone substrate,
with 18-20 % increase in conversion being observed (Table 2).
This is likely to be due to the increased stability of both the
ketone reduction, and the NADPH regeneration catalysts
present in the same reaction vessel over 24 h. The
enantioselectivity of the reaction was not affected by the
presence of the catalyst, in either free or bioconjugated form
(> 99 % ee (S) was obtained in all cases). Ketone reduction by
the artificial metalloenzymes was very slow, and could only be
detected at rhodium concentrations much higher than those
used in the regeneration experiments (TOF 0.1 - 0.2 h-1). This is
in line with previous reports, where a very low turnover was
observed with the [Cp*Rh(bipy)(H2O)]2+ catalyst, compared to
enzymatic ketone reduction, under aqueous conditions at
neutral pH. 8,42
oxidation) after 1 h heating was 4.95 ± 0.37 U mg-1
.
TbADH activity in the reduction direction remained relatively
constant, irrespective of the type of catalyst used. However,
when the incubation time was 24 h, the activity of TbADH
decreased drastically when free Rh catalyst was present, but
remained
> 80 % in the presence of the artificial
metalloenzymes (Figure 5). Previous work by Steckhan and co-
workers suggested no inhibition of TbADH after incubation
with 0.5 mM [Cp*Rh(bipy)(H2O)]Cl2, however their
experiments were performed at 30 ᵒC and did not state the
enzyme concentration.8 On the other hand, Hollmann and co-
workers have previously shown that inactivation of TADH by
the same catalyst occurred upon incubation with more than 4
equiv. of Rh. Moreover, inactivation was pH-dependent, with
80 % activity being retained after 1 h incubation at pH 8 and
room temperature, and only 25 % of the TADH activity
remaining after incubation at pH 6 with 100-fold Rh molar
excess.17 In our hands, increased inactivation of TbADH by the
Rh(III) complex can be observed at 50 ᵒC, but can be avoided
by physically separating the complex from the enzyme surface,
by encapsulation into the protein.
3 Experimental
3.1 Bioconjugation of metal complexes to TbADH
2.5 Cofactor recycling cascades
The gene encoding for wild-type TbADH bearing an N-terminal
StrepTagII, cloned in the expression vector pET21a, was
obtained from Biomatik (Ontario, Canada). Mutagenesis was
performed using the QuikChange Lightning Site-Directed
Encouraged by the beneficial effect of bioconjugation on
preventing mutual inactivation between TbADH and the Rh
regeneration catalysts, we developed an ADH – hybrid catalyst
cascade for the reduction of 4-phenyl-2-butanone. In this
system, TbADH was used as the same protein scaffold for both
ketone reduction and NADPH regeneration driven by formate
(see Figure 1).
Mutagenesis
Kit
(Agilent
Technologies),
following
manufacturer’s instructions. Recombinant overexpression was
carried out in E. coli BL21(DE3). Wild-type TbADH and mutants
6 | J. Name., 2012, 00, 1-3
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H59A-D150A-C203S-C283A-C295A (5M) and C203S-C283A- To test the inactivation of TbADH by the Rh catalyst, 1 μM
C295A (3M) were routinely characterized by SDS-PAGE, TbADH was incubated with 5 μM Rh catalyst (non-brominated
enzymatic activity, protein concentration, ICP-MS to determine catalyst, or corresponding artificial metalloenzyme) in 100 mM
metal content and Ellman assay to determine availability of sodium phosphate pH 7 at 50 °C. Samples were taken after 1h
thiol groups. The wild-type TbADH sequence, primers used for or 24h and the residual activity was assayed
mutagenesis and details of protein expression, purification and spectrophotometrically after the addition of 2-butanone (50
characterisation can be found in the Supporting Information. mM final concentration) and NADPH (0.2 mM final
Ligands Br-L1 and Br-L2, and metal complexes [Cp*Rh(Br- concentration).
L1)Cl)]Cl and [Cp*Rh(Br-L1)Cl)]Cl were synthesized according to
3.4 NADPH recycling assays
the literature.
Covalent conjugation of complexes [Cp*Rh(Br-L1)Cl)]Cl and The assay was performed by adapting previous literature
[Cp*Rh(Br-L1)Cl)]Cl to TbADH 5M was performed in 100 mM protocols.8 The reaction mixture (250 µL) contained 4-phenyl-
TrisHCl pH 7, by incubating 4-fold molar excess of the 2-butanone (5 mM), TbADH (1.5 μM), NADP+ (0.5 mM), Rh
brominated Rh catalysts with TbADH 5M, for 1 h at room catalyst (non-brominated catalyst, or corresponding artificial
temperature. Unbound catalyst was removed by desalting metalloenzyme, 25 μM) and sodium formate (500 mM), in 100
through
a PD-10 column (GE Healthcare), followed by mM sodium phosphate pH 7. The mixture was incubated for
concentration by ultrafiltration with Vivaspin 6 (10 kDa 24 h at 50 °C, then cooled to room temperature, extracted
MWCO, Sartorius). The flowthrough was routinely tested for with 1 volume of hexane, dried over Na2SO4 and analysed by
the absence of catalytic activity and Rh content, thus chiral HPLC. The analysis was performed with an Agilent 1220
supporting the absence of free catalyst in the artificial Infinity equipped with a CHIRALCEL® OD column (4.6mm x 250
metalloenzyme preparations. UV-visible absorbance spectra mm; particle size 10 μm). The separation was isocratic at 1 mL
were acquired in 100 mM sodium phosphate pH 7 with a Cary min-1, using an isopropanol / hexane mixture 6 / 94 (v / v). The
8454 spectophotometer (Agilent Technologies). Rh content detection wavelength was 215 nm. Quantification was
was determined by inductively coupled plasma mass achieved by comparing peak area against standard solutions.
spectrometry (ICP-MS) with
a Thermo Fisher iCAP-Q Omitting any of the component from the reaction mixture
instrument, after digestion of the protein in nitric acid (trace resulted in no conversion.
metal grade, Fisher Scientific) as previously described.43 Mass
spectrometry analysis was performed by electrospray
Conclusions
ionisation time-of-flight (ESI-TOF) in
a Bruker Impact II
spectrometer. Before analysis, the protein samples were
desalted in pure water, concentrated up to ~5 mg mL-1 and
then mixed with 1 volume of 0.1% formic acid in acetonitrile.
Alcohol dehydrogenase was for the first time used as a protein
host for the design and creation of artificial metalloenzymes.
Cp*Rh(III) complexes of bipyridine and phenanthroline bearing
an electrophilic moiety were covalently anchored to
thermostable TbADH devoid of active site zinc and containing a
single cysteine in the protein sequence. The resulting
conjugated complexes were shown to catalyse the reduction
of NADP+ at 50 °C. The mutual inactivation between wild-type
TbADH and the ADH-based artificial metalloenzymes was
evaluated and showed an increased stability of both the
rhodium catalysts and the wild-type TbADH, when
bioconjugated species were used instead of free complexes.
The ADH-based artificial metalloenzymes were successfully
combined in cofactor recycling cascades with wild-type TbADH,
and afforded higher conversions compared to the free
catalysts for the enantioselective reduction of 4-phenyl-2-
butanone. The activity of these artificial metalloenzymes
remains 2 orders of magnitude lower than the activity of
recently discovered NADP+-dependent native formate
dehydrogenase systems (0.02 s-1 vs up to 4.8 s-1).44 However,
their compatibility with high temperatures and neutral pH
makes them interesting candidates for further development of
regeneration methods with bioconjugated transition metal
complexes. Further research is in progress to optimise the
conjugation site and to improve the binding specificity of
rhodium complexes to TbADH. Additional metals such as Ru
and Ir, as well as different ligand architectures will also be
3.2 Formate-driven NAD(P)+ reduction assays
The reduction rate was measured by monitoring the
absorbance at 340 nm (ɛ = 6220 M-1 cm-1) with a Shimadzu UV-
2600 spectrophotometer, equipped with CPS-100 temperature
controller. Reactions were performed as previously described,7
in 100 mM sodium phosphate, 500 mM sodium formate pH 7
at 50 °C. NAD(P)+ concentration was 0.15 mM. Control
experiments were carried out in the absence of any catalyst or
in the absence of formate.
3.3 Stability assays
Metal concentration was determined by weight (free catalysts)
or by ICP-MS (artificial metalloenzyme). Stability tests were
adapted from previously reported mutual inactivation
experiments with [Cp*Rh(Bipy)H2O]2+ and TADH.17 To test the
inactivation of the Rh catalyst by TbADH, 10 μM Rh catalyst
(non-brominated complex, or corresponding artificial
metalloenzyme) was incubated with 10 μM TbADH in 100 mM
sodium phosphate pH 7 at 50°C. Samples were taken after 1h
or 24h and the residual activity was assayed
spectrophotometrically after the addition of sodium formate
(100 mM final concentration) and NADP+ (0.15 mM final
concentration).
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ARTICLE
Journal Name
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