A metal ion regulated artificial metalloenzyme

Article abstract of DOI:10.1039/c7dt00533d

Regulation of enzyme activity is essential in living cells. The rapidly increasing number of designer enzymes with new-to-nature activities makes it necessary to develop novel strategies for controlling their catalytic activity. Here we present the development of a metal ion regulated artificial metalloenzyme created by combining two anchoring strategies, covalent and supramolecular, for introducing a regulatory and a catalytic site, respectively. This artificial metalloenzyme is activated in the presence of Fe²⁺ ions, but only marginally in the presence of Zn²⁺.

Full text of DOI:10.1039/c7dt00533d

View Article Online
View Journal
Dalton
Transactions
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. Bersellini and
G. Roelfes, Dalton Trans., 2017, DOI: 10.1039/C7DT00533D.
Volume 45 Number
1
7
January 2016 Pages 1–398
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Dalton
Transactions
An international journal of inorganic chemistry
www.rsc.org/dalton
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

Page 1 of 6
Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Dalton
Transactions
View Article Online
DOI: 10.1039/C7DT00533D
PAPER
A metal ion regulated artificial metalloenzyme
Manuela Bersellini and Gerard Roelfes*
HoReceived 00th January 20xx,
Accepted 00th January 20xx
Regulation of enzyme activity is essential in living cells. The rapidly increasing number of designer enzymes with new-to-
nature activities makes it necessary to develop novel strategies for controlling their catalytic activity. Here we present the
development of a metal ion regulated artificial metalloenzyme created by combining two anchoring strategies, covalent
and supramolecular, for introducing a regulatory and a catalytic site, respectively. This artificial metalloenzyme is activated
DOI: 10.1039/x0xx00000x
www.rsc.org/
2+
2+
in presence of Fe ions, but only marginally in presence of Zn .
supramolecular
incorporation.
anchoring
and
unnatural
amino
acid
11–14
The resulting hybrid catalysts have been applied
Introduction
in a variety of catalytic reactions including enantioselective Diels
Alder, water addition to α,β-unsaturated ketones and Friedel-Crafts
alkylation reactions.
Control over enzymatic activity is vital for regulating the complex
network of biosynthetic pathways in living cells. An important
strategy employed by nature to regulate enzyme activity is
allosteric regulation, that is binding of an effector molecule or metal
ion at a site in the protein other than the active pocket. As a
result of this interaction, the catalytic activity of an enzyme and,
hence, its activity in a biosynthetic pathway can be regulated by the
presence or absence of chemical signals in the environment. Such
allosteric regulation would also be desirable for controlling the
activities of designer enzymes with new-to-nature activities.
Additionally, such artificial allosteric enzymes could also be of
interest for other applications, for example as sensors. In this work
we present a design of an artificial metalloenzyme that is selectively
For the creation of a metal regulated artificial metalloenzyme, we
took advantage of the design versatility of LmrR and combine two
anchoring strategies for binding transition metal complexes:
incorporation of a catalytic site via supramolecular interactions and
introduction of a regulatory site via covalent anchoring of a ligand.
The incorporation of the catalytic site via the supramolecular
1–3
2+
approach is based on the binding of Cu complexes of planar
aromatic ligands (such as phenanthroline) between the two
tryptophans located inside the hydrophobic pocket at the dimer
interface of LmrR at positions 96 and 96’ (Figure 1a). This approach
was applied to catalyze the enantioselective Friedel-Craft alkylation
2+
activated by a specific metal ion, that is Fe , but not by addition of
2+
of α,β-unsaturated imidazoles to indoles using Cu phenanthroline
2
+
Zn .
complexes, such as [Cu(phen)(NO ) ], obtaining good conversions
3
2
Artificial metalloenzymes are hybrid catalysts that incorporate non-
natural metal cofactors into biological scaffolds, thereby giving
access to enzyme-like catalysts with new-to-nature catalytic
14
and excellent enantioselectivities of >90% in several cases.
The introduction of the regulatory site via covalent anchoring of a
metal binding moiety to LmrR is based on the site-selective
4–7
activities. In recent years, this strategy has been utilized to create
a wide variety of artificial metalloenzymes that display non-natural
activities within a biological environment. However, examples of
such hybrid enzymes that can be activated by mechanisms akin to
allosteric control remain scarce. Indeed, the only example for this
type of activation comes from the Ward group, who has recently
demonstrated that the activity of an artificial asymmetric transfer
hydrogenase could be upregulated by proteolysis when a natural
modification of the protein using unique cysteines as
11,12
bioconjugation handles for alkylation reactions.
Since LmrR is a
homodimeric protein, the introduction of a ligand in a specific
position via covalent anchoring will lead to functionalization of both
monomers, resulting in a dimeric protein containing two metal
binding moieties.
8
protease is used as an external stimulus.
We have introduced a novel class of artificial metalloenzymes based
Results and discussion
on the transcriptional regulator LmrR (Lactococcal multidrug Our design of a metal ion regulated artificial metalloenzyme is
resistance Regulator). For this protein a new active site can be depicted in Figure 1b. In absence of an effector metal ion, the metal
created within the hydrophobic cavity that is present at the dimer binding moieties covalently bound to LmrR will bind the catalytically
9
,10
interface. An attractive feature of LmrR as biomolecular scaffold active complex [Cu(phen)(NO ) ]. As a result, the two free
3 2
2+
for artificial metalloenzymes is that it is the only scaffold reported coordination sites on the Cu ion required for substrate binding are
so far amenable to three different anchoring strategies for blocked, rendering the artificial metalloenzyme inactive.
incorporation
of
non-natural
cofactors:
covalent
and Conversely, addition of transition metal ions that bind stronger than
2+
Cu , would trap the metal binding moieties into stable chelate
2+
complexes, allowing the active Cu complex to bind and activate
the substrates (Figure 1b).
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands.E-mail: j.g.roelfes@rug.nl; Web: http://roelfesgroup.nl
Electronic Supplementary Information (ESI) available: [Full experimental details
and characterization, results of control and additional experiments]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Page 2 of 6
ARTICLE
a)
Journal Name
View Article Online
DOI: 10.1039/C7DT00533D
a)
b)
0
,12
0,08
,04
1
,5
0
,10
W96
W96'
0
0,08
0,06
1
,0
0
,15
,10
,05
0,00 400
0,00
0
20 40 60 80 100
2+
0
[Fe ] (mM)
0
,04
0
0
,5
b)
500
600
0,02
Wavelength (nm)
0
,00
0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8
2+
0,0
300
400
wavelength
500
nm
600
X
Fe
(
)
2
,0
0
,6
,4
,2
,0
0
0,5
0,4
0,3
1,5
0
0
0
20 40 60 80 100
2+
1,0
[Zn ] (mM)
0
,2
,1
,0
0
,5
0
0
0,0
0
,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8
300
400
wavelength (nm)
500
X Zn2+
Figure 3: a) UV-visible titrations of LmrR E104C_bpy with FeSO
4 2
·7H O (top) and
10
Zn(NO ) ·6H O (bottom). Titrations were performed in 50 mM NaH PO pH 7.0, 500
3
2
2
2
4
Figure 1: a) Surface representation of the front entrance of LmrR (PDB 3F8F). Zoom in
of part of the hydrophobic pocket at the dimer interface. Tryptophans in position 96
and 96’ are shown in blue. b) Schematic representation of the concept of the metal
regulated artificial metalloenzyme.
mM NaCl with 22.5 µM LmrR_bipyridine conjugate solutions (dimer) with subsequent
additions from 1 mM solutions of metal salts in milliQ water. Inset containing the fitting
of the titration data obtained with non-linear regression. b) Job’s plot graphs of LmrR
E104C_bpy with FeSO
4
·7H
for Fe and 311 nm for Zn against the molar fraction (x) of the Fe and Zn
respectively. 100 µM stock solutions of LmrR conjugates in 50 mM NaH PO pH 7.0, 500
2
O (top) and Zn(NO ) ·6H O (bottom). Plots of ΔA at 530 nm
3
2
2
2+
2
+
2
+
2
+
,
Previous work with LmrR based metalloenzymes showed that
2
4
enantioselective reactions take place inside the hydrophobic pocket mM NaCl and 200 µM of FeSO ·7H O or Zn(NO ) ·6H O in milliQ water. Total
4
2
3
2
2
in close proximity to the front entrance. Therefore it was envisioned concentration in the cuvette was kept constant at 60 µM.
that the formation of chelate metal complexes in this area might
11,12
Plasmids encoding for C-terminally Strep-tagged cysteines mutants
of LmrR (F93C, E104C, H86C) were prepared by standard site-
directed mutagenesis technique (Quick-Change) from a vector
also have an effect on the catalysis.
-2’-bipyridine was selected as metal binding moiety due to its
ability to strongly bind late-first row transition metal ions with high
2
11
15–19
available from previous work.
affinity and in different stoichiometries.
The bipyridine ligands
LmrR mutants were subsequently expressed in E.coli BL21 (DE3)
C43 and purified by affinity chromatography (Strep-tactin
Sepharose column) and cation exchange chromatography (Heparin
column) to remove residual bound DNA. Target proteins were
obtained in good yields (10-20 mg/L) with excellent purity as judged
by Tricine-SDS-PAGE, UPLC-MS and analytical size exclusion
chromatography (Figure S1).
were introduced on solvent exposed positions on the α4 helices,
which delimit the front entrance of the hydrophobic pocket.
Introduction of bipyridine ligands on these helices was envisioned
to allow the formation of chelate complexes involving one
bipyridine moiety of each monomer. Positions 93 and 104, located
in the central part of the front entrance, and position 86, which is
located towards the edge of the pocket, were chosen for the
introduction of the bipyridine ligands (Figure 2a).
Bipyridine units were introduced by selective alkylation of cysteines
with a bromo acetamide derivative of 2-2’-bipyridine (Figure
a)
11,12
2
b).
UPLC-MS and Tricine-SDS-PAGE were used to confirm
identity and purity of LmrR bipyridine conjugates and analytical size
exclusion chromatography demonstrated that the dimeric structure
of LmrR was not significantly altered by introduction of the metal
binding moieties (Figure S2-S4).
With the LmrR bipyridine conjugates in hand, first the binding of
2+
2+
these conjugates to Zn and Fe was studied by UV-visible
titrations. These two ions are examples of divalent late-first row
transition metal ions with different coordination properties. Upon
addition of either metal ion, the appearance of a shoulder around
b)
N
H
N
N
Br
N
O
H
SH
N
N
S
3
10 nm was observed, indicative of a change in the π–π* transition
5
0 mM NaH
50 mM NaCl
2
PO
4
pH 7.8
O
1
of the bipyridine upon metal coordination (Figure 3a, S5). This red
shift has previously been reported for bipyridine ligands in solution
and for proteins containing a bipyridine moiety.
4
°C, 8h, Ar
O
HS
S
18,22,23
N
N
H
In the
2+
N
titration with Fe also the appearance of two additional absorption
bands between 490 and 530 nm was observed, which are
17,19,24,25
2+
characteristic of Fe bipyridine complexes (Figure 3a, S5).
The resulting titration curves could be fitted to a 1:1 binding model,
Figure 2. a) Surface and helix representation of LmrR front entrance. Residues H86,
F93, E104 are shown in blue, green and red, respectively. b) Reaction scheme of the
synthesis of protein-metal binding moieties conjugates. Ligand used for alkylation corresponding to the formation of complexes containing one metal
reaction on LmrR cysteine mutants: N-([2,2’-bipyridin]-5-ylmethyl)-2-bromoacetamide
ion per LmrR dimer (Table S2). The stoichiometry of the
metal:protein complexes was further confirmed by Job’s plot
20,21
(1).
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 6
Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Journal Name
ARTICLE
2+
2+
Table 1 Results of vinylogous Friedel−Crafts alkylation reactions catalyzed by LmrR_bpy conjugates and their respective Fe and Zn complexes.
View Article Online
O
NO
mM)
O
DOI: 10.1039/C7DT00533D
[
Cu(phen)(
LmrR (30 mM)
3
)
2
] (22
N
+
N
*
2
0 mM MOPS, 500 mM NaCl
N
4ºC, 72h
N
pH=7.0,
N
H
NH
1
2
3
a
b
c
Entry
Catalyst
Yield (%)
TON
ee (%)
1
2
3
4
5
6
7
8
9
Cu(phen)(NO
3
)
2
(only)
39±4
58±3
≤1
16±2
24±1
-
<5
wt-LmrR
92±1
n.d.
LmrR F93C_bpy
LmrR E104C_bpy
LmrR H86C _bpy
≤1
-
n.d.
7±1
2±0
2±1
34±4
4±0
26±6
14±2
3±1
1±0
1±1
14±2
2±0
11±2
6±1
81±4
n.d.
2+
LmrR F93C _bpy+Fe
2+
LmrR F93C _bpy+Zn
n.d.
2+
LmrR E104C_bpy+Fe
75±6
n.d.
2+
LmrR E104C_bpy+Zn
2+
10
LmrR H86C _bpy+Fe
88±3
2+
11
LmrR H86C_bpy+Zn
80±10
3 2
Typical conditions: 2.2 mol% [Cu(phen)(NO ) ] (22 µM) loading with 1.3 eq LmrR_bpy conjugates (30 µM), 1 mM of substrate 1 and 2 in 20 mM MOPS buffer pH 7.0, 500 mMa
NaCl, for 72 h at 4 °C. All the results listed correspond to the average of two independent experiments, each carried out in duplicate. Errors are listed as standard deviations.
b
Yields were determined by HPLC and using 2-phenylquinoline as internal standard. Turnover numbers (TON) were determined by dividing the concentration of product by
c
the catalyst concentration. For yields below 5% ee’s were not determined.
(
Figure 3b, S6), in which the maximum complex formation was incubation of 1 equivalent of the corresponding metal salt with 1
obtained with equimolar concentrations of metal ions and LmrR equivalent of the LmrR_bpy conjugate (dimer) in 20 mM MOPS, pH
dimer. ICP-AES experiments performed after incubation of the 7.0, 500 mM NaCl (4°C, 30 minutes). The mixtures were centrifuged
2+
2+
LmrR_E104C_bipyridine conjugate with Fe and Zn , followed by to remove possible precipitate and the supernatant containing the
2+
dialysis to remove unbound metal ions, also suggested the presence LmrR_bipyridine_M complex was used for the supramolecular
of one equivalent of metal ion per LmrR dimer (Table S4). assembly of the artificial metalloenzyme. The artificial
Combined these observations suggest the formation of a chelate metalloenzymes were prepared in situ by self-assembly from
metal complex between one metal ion and two bipyridines in the [Cu(phen)(NO ) ] (22 μM) with a slight excess (1.3 equivalents) of
3
2
2+
LmrR scaffold. Exogenous water molecules or ligands supplied by LmrR_bipyridine_M complex (30 μM) in 20 mM MOPS, pH 7.0,
amino acid side chains might complete the coordination sphere 500 mM NaCl. The mixture was incubated at 4°C for 30 minutes and
around the metal. The formation of a chelate complex between the the catalytic reaction was initiated by addition of substrates 1 and 2
two bipyridines and the metal ions was expected for the conjugates at a final concentration of 1 mM. Reactions were incubated at 4⁰C
containing bipyridine units located in the central part of the front for 72 h, after which the products were isolated and analyzed by
entrance (LmrR F93C_bpy and LmrR E104C_bpy), given the chiral HPLC.
proximity of the two amino acids in the protein dimer (10-15 Å from As previously reported, the reaction of the α-β unsaturated acyl
10
the reported crystal structure). However, for the conjugate in imidazole 1 with 2-methyl indole 2 catalyzed by the assembly of
which the bipyridine units are located towards the edge of the [Cu(phen)(NO ) ] with wt-LmrR is protein accelerated and results in
3
2
14
hydrophobic pocket (LmrR H86C_bpy) the formation of a chelate excellent enantioselectivity up to 92% (Table 1, entries 1, 2). As
metal complex appeared unlikely due to the distance between the expected, using the LmrR_bipyridine conjugates in absence of any
10
two residues in position 86 (28-30 Å). Tentatively, chelation of the regulatory metal ion resulted in no significant catalytic activity of
metal ions could be achieved due to the known flexibility of the the artificial metalloenzymes with LmrR F93C_bpy and LmrR
10
LmrR scaffold. The cost for formation of these rather unfavoured E104C_bpy, and low activity in case of LmrR H86C_bpy (Table 1,
complexes is reflected in a lower binding affinity for both regulatory entries 3-5). This is due to the fact that the free coordination sites
2+
metals complexes (Table S2) and an apparent lower stability of the of the Cu ion in the [Cu(phen)(NO ) ] complex are sequestered by
3
2
metal-bound protein, which was prone to precipitation. However, the conjugated bipyridine ligands, thus preventing substrate
also the possibility of formation of chelate metal complexes with binding (Figure 1b). In case of LmrR H86C_bpy, this sequestration is
bipyiridine units from separate LmrR dimers should be considered, apparently not perfect, for reasons not understood at present.
2+
2+
as suggested by the presence of higher order aggregates in the size Formation of metal complexes with either Fe or Zn prior to
2+
exclusion chromatography of some Fe
E104C_bpy and LmrR H86C_bpy) (Figure S4).
conjugates (LmrR catalysis, resulted in almost no product formation for the conjugate
LmrR F93C_bpy (Table 1, entries 6, 7). The observed lack of catalytic
The catalytic activity of the LmrR_bipyridine conjugates and their activity might be due to bulkiness of the chelate metal complexes
metal complexes, was evaluated in the enantioselective, vinylogous located right in the middle of the front entrance of the hydrophobic
2
+
Friedel−Crafts alkylation of 1-(1-methyl-1H-imidazol-2-yl)but-2-en- pocket that does not allow the substrates to reach the active Cu
13,14,26–29
1
-one (1) with 2-methyl-1H-indole (2).
center located inside. This hypothesis is supported by the
The metal complexes of LmrR bipyridine conjugates with Fe and observation that no specific binding of the dye Hoechst 33342
Zn (LmrR_bpy_M ) were prepared fresh prior to catalysis by (H33342), which is known to bind inside the hydrophobic pocket of
2+
2+
2+
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Page 4 of 6
ARTICLE
Journal Name
LmrR via the front entrance, to LmrR F93C_bpy was observed using to the second bipyridine in the LmrR scaffold (Figure 5Vaie)w. IAnrtoicrledOenrlitnoe
1
0
fluorescence spectroscopy (Figure S7).
push the equilibrium towards a compleDteOI:m10e.t1a0l39r/eCp7lDaTce0m05e3n3Dt,
2+
We were pleased to find that upon incubation with the regulatory leading to a Cu bisbipyridine complex and the release of the
metal ions (Fe or Zn ) prior to addition of the active regulatory metal ion, incubation of the LmrR_bpy_M complexes
2+
2+
2+
[
Cu(phen)(NO ) ] complex, two of conjugates, i.e. LmrR E104C_bpy with Cu(NO ) was performed (Figure 5b).
3
2
3
2
and LmrR H86C_bpy, showed catalytic activity (Table 1, entries 8-
0), albeit lower than with wt-LmrR. These results suggest that the
2+
2+
LmrR_bpy_E104C_bpy_Fe
LmrR_bpy_E104C_bpy_Zn
1
30
25
100
80
activity of these artificial metalloenzymes can be regulated by the
presence of metal ions.
2
0
6
0
2+
15
It was observed consistently that activation with Fe yielded more
active artificial metalloenzymes when compared to the addition of
Zn : higher turnover numbers (TON) were achieved for the artificial
metalloenzymes containing Fe complexes compared to the
respective Zn complexes (Table 1, entries 8-9, 10-11). It was Figure 4: Comparison of Turnover Numbers (black) and enantioselectivity (purple) in
hypothesized that the difference in activity might reflect the
different interactions of the active transition metal complex
(
40
10
2+
20
0
5
0
2+
0 eq
1eq
3eq
6eq
2+
the vinylogous Friedel−Crafts alkylation reactions catalyzed by LmrR_bpy_E104C_bpy
2+
2+
conjugate and respective Fe and Zn complexes in presence of increasing
2+
2+
concentration of Fe (left) and Zn (right). Same reaction conditions as in Table 1.
2+
[Cu(phen)(NO ) ]) with the two types of metal complexes (Fe and
3
2
2+
2+
Zn
complex). Nevertheless, as described above, the Fe
a)
complexes of these two conjugates, in addition the dimeric form of
LmrR, appeared to form higher order aggregates as well. At this
moment it is not possible to assess whether these aggregates have
some influence on not clear what effect these aggregates have on
catalysis.
As this difference in activity is particularly pronounced for the LmrR
E104C_bpyridine conjugate, this variant was selected for further
investigation. According to the Irving-Williams series, six-coordinate
b)
2+
octahedral Cu complexes are thermodynamically more stable than
other high spin divalent first-row transition metal complexes.
2
+
Hence, in our design, the Cu from the active [Cu(phen)(NO ) ]
3
2
Figure 5: Schematic representation of metal ion exchange between regulatory metal
2
+
2+
2+
complex could replace Zn
from the high spin bipyridine ion (M ) and the catalytic metal ion (Cu ) with [Cu(phen)(NO ) ] and Cu(NO ) .
3
2
3 2
complexes, forming a mixed complex with phenanthroline and the
covalently bound bipyridine as ligands. As this additional interaction
would prevent substrate binding, the resulting artificial
metalloenzyme becomes inactive for the desired reaction.
2
+
2+
Quantification of the regulatory metal (Fe or Zn ) bound to the
protein after incubation with Cu(NO ) showed a significant
3
2
2+
decrease in the amount of Zn bound to the protein (Table S3,
Figure S8), confirming the hypothesis of partial replacement of Zn
with Cu with subsequent inactivation of the catalyst. The
observation that the metal displacement reaction does not seem to
2+
2+
Conversely, Fe is known to predominantly form low spin
complexes with bipyridine ligands, which are kinetically more stable
and, thus, less prone to ligand substitution. Therefore, exchange of
2+
2+
2+
2+
happen for the Fe complex supports the formation of a stable low
Fe by Cu will not occur and the artificial metalloenzyme will
2
+
3
1
3
0,31
spin Fe bisbipyridine complex within the LmrR scaffold.
remain active.
To test this hypothesis, reactions were performed using an excess
Finally the reversibility of the designed metal regulated artificial
metalloenzyme was tested by inverting the order of addition of the
regulatory and catalytic metal ions. LmrR_bipyridine conjugates
were first incubated with [Cu(phen)(NO ) ] at 4°C for 30 minutes
2+
2+
of Fe or Zn ions. The LmrR_E104C_bpy conjugates were
incubated with 1, 3 and 6 equivalents of metal ions and in case of
2+
3
2
Zn also 10 equivalents (with respect to LmrR dimer) before the
artificial metalloenzyme was assembled by addition of
2
+
2+
and then 1 equivalent of Fe or Zn salt was added. After
incubation of the resulting mixture for 30 minutes substrates 1 and
were added at a final concentration of 1 mM. In parallel, exactly
the same procedure was applied to the LmrR_E104C_bpy
2+
[
Cu(phen)(NO ) ]. As expected, when additional Fe equivalents
3 2
2
were added, no significant change in catalytic activity was observed.
The possibility of metal exchange between the regulatory metal ion
2+
2
+
2+
2+
conjugates by incubating first the regulatory metal ions (Fe or
2+
(
Fe or Zn ) and the catalytic metal ion (Cu ) was further
2+
Zn ) and then the active catalyst [Cu(phen)(NO ) ]. Reactions were
3 2
investigated by ICP-AES measurements. LmrR_E104C_bpy_ M
complexes were incubated with Cu ions, followed by dialysis to
remove non-bound metal ions.
Quantification of the regulatory metal (Fe or Zn ) bound to the
protein after incubation with [Cu(phen)(NO ) ] did not show a
change in the total amount of Fe or Zn (Table S3) because metal
2+
incubated at 4⁰C for 72 h, after which the products were isolated
and analyzed by chiral HPLC. We were delighted to observe that
catalytic activity (as well as enantioselectivity) could be restored to
2+
2+
2
+
a large extent upon addition of Fe salt to the inactive
3
2
2+
2+
2+
metalloenzyme. In contrast, addition of Zn salt did not restore the
2
+
activity. This result suggests that selective regulation of activity of
exchange will result in the formation a mixed Cu phenanthroline
bipyridine complex, while the regulatory metal might still be bound
2
+
the designed artificial metalloenzyme by Fe ions is reversible.
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 6
Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Journal Name
ARTICLE
View Article Online
Table 2 Effect of the order of incubation on the results of vinylogous Friedel−Crafts alkylation reactions catalyzed by the [Cu(phen)(NO
3
)
2
]D O/ IL : m1 r0R . _1 0b p3 y9 _/ EC1 70 D4 CT _0b 0p 5y 33D
conjugate.
st
nd
a
b
c
Entry
1
incubation
2
incubation
-
Yield (%)
TON
ee (%)
n.d.
1
2
[Cu(phen)(NO
3
)
2
]
≤1
≤1
2+
Fe
[Cu(phen)(NO
3
)
2
]
]
57±14
23±5
14±2
6±1
72±14
78±2
2+
3
4
5
Cu(phen)(NO
3
)
2
]
Fe
[Cu(phen)(NO
33±5
13±2
11±3
2+
Zn
3
)
2
50±1
2+
[Cu(phen)(NO
3
)
2
]
Zn
5±1
50±13
3 2
Typical conditions: 2.2 mol% [Cu(phen)(NO ) ] (22 µM) loading with 1.3 eq LmrR_bpy conjugates (30 µM), 1 mM of substrates 1 and 2 in 20 mM MOPS buffer pH 7.0, 500
mM NaCl, for 72 h at 4 °C. All the results listed correspond to the average of two independent experiments, each carried out in duplicate. Errors are listed as standard
a
b
deviations.. Yields were determined by HPLC and using 2-phenylquinoline as internal standard. Turnover numbers (TON) were determined by dividing the concentration
c
of product by the catalyst concentration. For yields below 5% ee’s were not determined.
1
0 P. K. Madoori, H. Agustiandari, A. J. M. Driessen and A.-M. W. H.
Thunnissen, EMBO J., 2009, 28, 156–166.
Conclusions
11 J. Bos, F. Fusetti, A. J. M. Driessen and G. Roelfes, Angew. Chem.
Int. Ed., 2012, 51, 7472–7475.
The results presented here show that we successfully designed,
synthesized and characterized a metal ion regulated artificial
metalloenzyme. By combining a regulatory site to bind an effector
metal ion and an active site to recruit a catalytically active metal
complex in the LmrR scaffold, the activity of the artificial
metalloenzyme (LmrR_E104C_bpy) for a vinylogous Friedel−Crafts
1
1
1
1
2 J. Bos, A. García-Herraiz and G. Roelfes, Chem. Sci., 2013, 4,
578–3582.
3 I. Drienovská, A. Rioz-Martínez, A. Draksharapu and G. Roelfes,
Chem. Sci., 2014, 6, 770–776.
4 J. Bos, W. R. Browne, A. J. M. Driessen and G. Roelfes, J. Am.
Chem. Soc., 2015, 137, 9796–9799.
5 G. Atkinson and J. E. Bauman, Inorg. Chem., 1962, 1, 900–904.
3
2+
alkylation could be regulated by incubation with Fe ions.
Reminiscent of allosteric regulation in natural enzymes, we
2+
2+
16 R. H. Holyer, C. D. Hubbard, S. F. A. Kettle and R. G. Wilkins,
achieved selective activation by Fe but not by Zn , taking
advantage of the different coordination properties of these
transition metals. This study represents the first example of a metal
ion regulated artificial metalloenzyme and presents a significant
advance toward controlling the activity of designer enzymes in
hybrid bio-synthetic pathways.
Inorg. Chem., 1965, 4, 929–935.
1
1
7 P. Krumholz, J. Am. Chem. Soc., 1949, 71, 3654–3656.
8 R. M. Franzini, R. M. Watson, G. K. Patra, R. M. Breece, D. L.
Tierney, M. P. Hendrich and C. Achim, Inorg. Chem., 2006, 45,
9
798–9811.
9 M. Kurihara, K. Ozutsumi and T. Kawashima, J. Solut. Chem., 24,
19–734.
1
2
7
0 A. Basnet, P. Thapa, R. Karki, Y. Na, Y. Jahng, B.-S. Jeong, T. C.
Jeong, C.-S. Lee and E.-S. Lee, Bioorg. Med. Chem., 2007, 15,
4351–4359.
Acknowledgements
This work was supported by European Research Council (ERC
Starting Grant 280010). Financial support from the Ministry of 21 R. Ballardini, V. Balzani, M. Clemente-León, A. Credi, M. T.
Education, Culture, and Science (Gravitation Program No.
24.001.035) is gratefully acknowledged. The authors thank I.
Gandolfi, E. Ishow, J. Perkins, J. F. Stoddart, H.-R. Tseng and S.
Wenger, J. Am. Chem. Soc., 2002, 124, 12786–12795.
0
Drienovská and Dr L. Villarino for useful discussion and suggestions.
22 J. Xie, W. Liu and P. G. Schultz, Angew. Chem. Int. Ed., 2007, 46,
9
239–9242.
2
3 J. H. Mills, S. D. Khare, J. M. Bolduc, F. Forouhar, V. K. Mulligan,
S. Lew, J. Seetharaman, L. Tong, B. L. Stoddard and D. Baker, J.
Am. Chem. Soc., 2013, 135, 13393–13399.
Notes and references
1
N. M. Goodey and S. J. Benkovic, Nat. Chem. Biol., 2008, 4, 474–
82.
Q. Cui and M. Karplus, Protein Sci., 2008, 17, 1295–1307.
J. Fastrez, ChemBioChem, 2009, 10, 2824–2835.
F. Rosati and G. Roelfes, ChemCatChem, 2010, 2, 916–927.
J. Bos and G. Roelfes, Curr. Opin. Chem. Biol., 2014, 19, 135–143.
J. C. Lewis, Curr. Opin. Chem. Biol., 2015, 25, 27–35.
2
4 K. D. Barker, A. L. Eckermann, M. H. Sazinsky, M. R. Hartings, C.
Abajian, D. Georganopoulou, M. A. Ratner, A. C. Rosenzweig and
T. J. Meade, Bioconjug. Chem., 2009, 20, 1930–1939.
5 H. K. Munch, S. T. Heide, N. J. Christensen, T. Hoeg-Jensen, P. W.
Thulstrup and K. J. Jensen, Chem. – Eur. J., 2011, 17, 7198–7204.
6 A. J. Boersma, B. L. Feringa and G. Roelfes, Angew. Chem. Int.
Ed., 2009, 48, 3346–3348.
7 D. A. Evans, K. R. Fandrick, H.-J. Song, K. A. Scheidt and R. Xu, J.
Am. Chem. Soc., 2007, 129, 10029–10041.
8 D. A. Evans, K. R. Fandrick and H.-J. Song, J. Am. Chem. Soc.,
4
2
3
4
5
6
7
2
2
2
2
2
3
E. M. Brustad and F. H. Arnold, Curr. Opin. Chem. Biol., 2011, 15,
2
01–210.
8
Z. Liu, V. Lebrun, T. Kitanosono, H. Mallin, V. Köhler, D.
Häussinger, D. Hilvert, S. Kobayashi and T. R. Ward, Angew.
Chem. Int. Ed., 2016, 55, 11587–11590.
2
005, 127, 8942–8943.
9 T. B. Poulsen and K. A. Jørgensen, Chem. Rev., 2008, 108, 2903–
915.
0 M. A. Halcrow, Crystals, 2016, 6, 58.
9
J. Lubelski, A. De Jong, R. Van Merkerk, H. Agustiandari, O. P.
Kuipers, Jan Kok and A. J. M. Driessen, Mol. Microbiol., 2006, 61,
2
7
71–781.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Ple aDs ea l dt oo nn To rt a and sj ua sc tt imo na sr gins
Page 6 of 6
ARTICLE
Journal Name
3
1 F. H. Burstall and R. S. Nyholm, J. Chem. Soc. Resumed, 1952,
570–3579.
View Article Online
DOI: 10.1039/C7DT00533D
3
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins