Cross-Regulation of an Artificial Metalloenzyme

Article abstract of DOI:10.1002/anie.201702181

Cross-regulation of complex biochemical reaction networks is an essential feature of living systems. In a biomimetic spirit, we report on our efforts to program the temporal activation of an artificial metalloenzyme via cross-regulation by a natural enzyme. In the presence of urea, urease slowly releases ammonia that reversibly inhibits an artificial transfer hydrogenase. Addition of an acid, which acts as fuel, allows to maintain the system out of equilibrium.

Full text of DOI:10.1002/anie.201702181

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Cross-Regulation of an Artificial Metalloenzyme
Authors: Yasunori Okamoto and Thomas R. Ward
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201702181
Angew. Chem. 10.1002/ange.201702181
Link to VoR: http://dx.doi.org/10.1002/anie.201702181
http://dx.doi.org/10.1002/ange.201702181

10.1002/anie.201702181
Angewandte Chemie International Edition
COMMUNICATION
Cross-Regulation of an Artificial Metalloenzyme
Yasunori Okamoto,[a] and Thomas R. Ward*[a]
Dedicated to Prof. Roald Hoffmann on the occasion of his 80th birthday
Abstract: Cross-regulation of complex biochemical reaction
networks is an essential feature of living systems. In a biomimetic
spirit, we report on our efforts to program the temporal activation of
an artificial metalloenzyme via cross-regulation by a natural enzyme.
In the presence of urea, urease slowly releases ammonia that
reversibly inhibits an artificial transfer hydrogenase. Addition of an
acid, which acts as fuel, allows to maintain the system out of
equilibrium.
Thanks to numerous attractive features, biocatalysis, relying on
isolated enzymes, has gained increasing attention among the
synthetic chemistry community.[1] To further improve on this
asset, the focus is shifting towards enzyme cascades whereby
an intermediate produced by a first enzyme acts as the
substrate for a second enzyme etc. Such in-series assembled
enzymes (Scheme 1a) allow to circumvent the time-consuming
isolation and purification of intermediates. However, increasing
the complexity of enzyme cascades often lead to suboptimal
material flux as well as to inhibition.[2]
In living systems operating far from equilibrium, biochemical
cascades have evolved to include signal transduction as well as
metabolic- and allosteric regulation safeguards that affect both
the spatial control of intermediates and the temporal occurrence
of events.[3]
Thanks to the progress in computational systems biology and
genetic engineering, it has become possible to engineer in vivo
synthetic gene networks to control some of the fundamental
properties of living systems.[4] However, understanding at the
molecular level the details of such complex, off-equilibrium
(bio)chemical reaction networks remains challenging. [5-8]
In the past decade, artificial metalloenzymes (ArMs) have
attracted increasing attention as alternative to both
homogeneous
metalloenzymes result from the incorporation of an abiotic metal
cofactor within protein environment. In contrast to
catalysts
and
enzymes.[9]
Artificial
a
homogeneous catalysts, ArMs can be combined with natural
enzymes to afford enzyme cascades.[10] With the long-term goal
of engineering artificial metabolic pathways,[11] we report on our
efforts to introduce a programmable enzymatic temporal control
of the activity of an artificial metalloenzyme.
Scheme 1. Enzyme cascades, combined in series a) and in parallel b);
phosphofructokinase, a key committed step of glycolysis, is inhibited by citrate.
(ArM is an artificial metalloenzyme) c).
In order to program such an off-equilibrium control of the activity
of an ArM, we reasoned that one could combine in parallel an
enzyme that produces a reversible inhibitor of the ArM, Scheme
1b. Such cross-regulated biochemical networks play a key role
in living systems, Scheme 1c. With only starting materials
present at the onset of the reaction, both the enzyme and the
ArM are active. As the product of both enzymatic reactions
accumulate, the ArM is inactivated, Scheme 1b. Ideally, upon
removal or consumption of the inhibitor, the activity of the ArM is
restored. Building on our previous experience with artificial
transfer hydrogenases (ATHase hereafter) based on the biotin-
[a]
Dr. Y. Okamoto, Prof. Dr. T. R. Ward
Department of Chemistry
University of Basel
Spitalstrasse 51, CH-4056 Basel Switzerland
E-mail: thomas.ward@unibas.ch
Supporting information for this article is provided via a link at the end
of the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201702181
Angewandte Chemie International Edition
COMMUNICATION
avidin technology,[9b] we set out to program the activity ATHase
by combining it with an enzyme that produces an inhibitor.
In order to conveniently monitor the activity in real-time, we
selected enrofloxacin
1
as an ATHase substrate.[12] The
fluoroquinolone substrate 1 is a versatile antibiotic against both
gram-positive and gram-negative bacteria. Upon reduction of the
C=C double bond, the resulting β-ketoacid spontaneously
decarboxylates to afford a yellow product 2 (Figure S1). The
carboxylate group of enrofloxacin
1 is essential for its
antibacterial activity; its loss results in a significant increase of
minimal inhibitory concentration.[13]
We selected an ATHase comprised of a biotinylated Cp*Ir(4,7-
dihydroxy-1,10-phenanthroline) 3 and the streptavidin K121R
variant (Sav K121R). The ArM [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R
was previously reported for its remarkable ATHase activity for
the reduction of imines, using either NAD(P)H or formate as
hydride source in the presence of a variety of enzymes, sharing
a
common intermediate (i.e. in-series enzyme cascades,
Scheme 1 a).[10c] A preliminary screen led to the identification of
the following reaction conditions: ATHase (10 µM, 0.5 mol%)
enrofloxacin 1 (2 mM), formate (2M) and MOPS (0.1 M, pH 7.0).
At 37 ºC, 96 turnovers (96 TON) were obtained after 8 hours.
Next, the pH-dependence of the [(Biot-Cp*)IrCl(N^N)] 3 · Sav
K121R activity was evaluated by determining the initial rate of
the reduction of enrofloxacin 1 by monitoring the appearance of
the ketone 2 at 430 nm (Scheme 2). A bell-shape pH-profile
was observed, both for the free non-biotinylated cofactor
[(Cp*)IrCl(N^N)] 4 as well as the ATHase [(Biot-Cp*)IrCl(N^N)] 3·
Sav K121R. As the ATHase [(Biot-Cp*)Ir(N^N)] 3· Sav K121R is
completely, but reversibly inhibited at pH > 8.0, we speculated
that we may cross-regulate its activity by coupling it in parallel
with an enzyme that produces a base as reaction product,
Scheme 1b. To evaluate the stability of the ATHase upon cycling
the pH from pH < 6.0 to pH > 8.5, the reaction mixture was
repeatedly spiked with HCl and NaOH. Monitoring the
absorbance at 430 nm unambiguously demonstrates the activity
of the ArM towards enrofloxacin 1 in acidic medium. Addition of
NaOH causes the ATHase to stall. Subsequent addition of HCl
leads to a 30% erosion of ATHase activity for the second cycle.
Gratifyingly, the activity for the second to fifth cycles remains
constant, Scheme 2c and Figure S2.
Scheme 2. Structure of the biotinylated iridium complex 3 and schematic
representation of the corresponding artificial transfer hydrogenase (ATHase)
tested for the reduction of enrofloxacin 1 using formate as hydride source a).
pH-dependence of enrofloxacin 1 reduction by ATHase [(Biot-Cp*)Ir(N^N)] 3 ·
Sav K121R (solid line) and by [(Biot-Cp*)Ir(N^N)] 4 (dashed line) b). Activity
profile resulting from pH cycling upon addition of HCl and NaOH (see also
Figure S2) c). Reaction conditions: 10 µM ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav
K121R (or complex 4), 1 mM enrofloxacin 1, and 2 M HCOONa in 10 mM
buffer at 37 ºC.
Urease from Canavalia ensiformis (Jack bean, E.C. 3.5.1.5) was
selected as alkali-generating enzyme to be integrated in parallel
with the ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R. Urea rapidly
reacts with urease to afford carbon dioxide and two equivalents
of ammonia. Addition of bromothymol blue allows to
conveniently monitor the pH profile (i.e. reflecting the urease
activity) by relying on the absorption ratio at 618 nm/501 nm
(absorption maximum and isosbestic point respectively) (Figure
S3). In the presence of either [(Cp*)IrCl(N^N)] 4 or [(Biot-
Cp*)IrCl(N^N)] 3, no pH variation could be observed upon
addition of urease and urea (Scheme S1). We conclude that
This article is protected by copyright. All rights reserved.

10.1002/anie.201702181
Angewandte Chemie International Edition
COMMUNICATION
either organometallic catalyst [(Cp*)IrCl(N^N)] 4 or bare cofactor
[(Biot-Cp*)IrN^N] 3 inactivates the urease. In the presence of
both the ATHase [(Biot-Cp*)IrN^N] 3 · Sav K121R and urease,
the pH gradually increases upon addition of urea, highlighting
the compatibility of both ArM and urease partners (Scheme S2).
Upon lowering the pH to 5.0 by addition of HClaq, both the
ATHase and the urease are activated as reflected by the
appearance of product 2 and the slow increase in pH. The
ATHase remains active until pH = 8 at which point, it is inhibited
by the product of urease: ammonia, Scheme 3b. Addition of
HClaq, restores 86 % of the initial ATHase’s activity, highlighting
the reversible inhibition of the ATHase, Scheme 3c. As can be
appreciated, the activity of the ATHase (as revealed by the
increase in absorbance at 430 nm) correlates with the pH
variation (monitored by the ratio of absorbance at 618 nm/501
nm in the presence of bromothymol blue as pH indicator),
Scheme 3c, Figure S4 and Scheme S4. In the presence of
urease, the ATHase stalls below pH 7.0, instead of pH > 8.0 for
the isolated ATHase (Scheme 2b). We speculate that this arises
from the formation of ammonia from urea which competes with
formate as a ligand for the Ir-center thus leading to an earlier
onset of (reversible) inhibition. Upon fine-tuning the
concentrations of urea and the urease, the activity window of the
ATHase can be time-programmed, Scheme 3b.
Enzymatic logic gates producing a pH change as output have
been reported. In such systems, urease and esterase are used
to produce base and acid respectively.[14] Building upon this, we
hypothesized that one could use
a dormant activator to
substitute HCl, and thus program the progressive onset of
ATHase activity. We evaluated the performance of the ATHase
[(Biot-Cp*)Ir(N^N)] 3 · Sav K121R in the presence of urease, that
produces the alkali inhibitor and the esterase from porcine liver
(E.C. 3.1.1.1) and its substrate ethyl butyrate, the dormant
activator, Scheme 4. Upon addition of ethylbutyrate to a solution
containing urea (the dormant deactivator), enrofloxacin 1, the
ATHase, urease and the esterase, a slow onset of ATHase
activity was observed as well as a slow inhibition resulting from
the production of ammonia. In the absence of esterase, no
product 2 could be detected.
Scheme 3. Schematic representation of the pH-dependent activation of
ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R and urease a). Time-dependent
reduction of enrofloxacin 1 by the ATHase in the presence of urease and urea.
The reaction was monitored at 430 nm (See Figure S1) b). (Re)activation of
the ATHase (blue trace) and urease by addition of acid (orange trace) c).
Experimental conditions; 25 µM ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R, 1
mM enrofloxacin 1, 0.1 – 0.4 mg/ml urease, 150 – 300 mM urea and 2 M
HCOONa in 10 mM CHES (pH 9.0) at 37 ºC. An aliquot of 5 M HCl (3.2 µl and
5 µl for 1st and 2nd addition respectively to bring the pH down to 5.0) was
added per 200 µl of the reaction mixture (see also Scheme S3).
Next, the activity of ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R
was evaluated in the presence of urease and urea. The initial pH
was set to pH 9.0, where both urease and ATHase are inactive.
This article is protected by copyright. All rights reserved.

10.1002/anie.201702181
Angewandte Chemie International Edition
COMMUNICATION
Acknowledgements
Generous support from the Swiss National Science Foundation
(grant 200020_162348) and from the NCCR Molecular Systems
Engineering, an advanced ERC grant (DrEAM agreement
number 694424) as well as
a JSPS Overseas research
fellowship to YO is gratefully acknowledged. We thank Dr. M.
Dürrenberger, Dr. V. Köhler and Dr. J. Rebelein for enlightening
discussions and Umicore for a loan of [Cp*IrCl2]2.
Keywords: artificial metalloenzyme • cross-regulation • urease •
hydrogenation • biocatalysis
[1]
a) A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B.
Witholt, Nature 2001, 409, 258-268; b) A. J. Straathof, S. Panke, A.
Schmid, Curr. Opin. Biotechnol. 2002, 13, 548-556; c) R. N. Patel, ACS
Catal. 2011, 1, 1056-1074; d) J. H. Schrittwieser, J. Sattler, V. Resch, F.
G. Mutti, W. Kroutil, Curr. Opin. Chem. Biol. 2011, 15, 249-256; e) U. T.
Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K.
Robins, Nature 2012, 485, 185-194; f) M. T. Reetz, J. Am. Chem. Soc.
2013, 135, 12480-12496; g) J. A. Rollin, T. K. Tam, Y. H. P. Zhang,
Green Chem. 2013, 15, 1708-1719; h) N. J. Turner, M. D. Truppo, Curr.
Opin. Chem. Biol. 2013, 17, 212-214.
[2]
a) M. Bujara, M. Schumperli, R. Pellaux, M. Heinemann, S. Panke, Nat.
Chem. Biol. 2011, 7, 271-277; b) X. Chen, C. Zhang, R. Zou, K. Zhou,
G. Stephanopoulos, H. P. Too, PLoS One 2013, 8, e79650; c) C. Hold,
S. Billerbeck, S. Panke, Nat. Commun. 2016, 7, 12971.
[3]
[4]
a) D. E. Koshland, Jr., A. Goldbeter, J. B. Stock, Science 1982, 217,
220-225; b) D. Bray, Nature 1995, 376, 307-312; c) B. N. Kholodenko,
Nat. Rev. Mol. Cell Biol. 2006, 7, 165-176; d) J. E. Ferrell, Jr., T. Y. Tsai,
Q. Yang, Cell 2011, 144, 874-885.
a) B. Novak, J. J. Tyson, Nat. Rev. Mol. Cell Biol. 2008, 9, 981-991; b)
J. Kim, E. Winfree, Mol. Syst. Biol. 2011, 7, 465; c) J. Hasty, D.
McMillen, J. J. Collins, Nature 2002, 420, 224-230; d) R. Kojima, D.
Aubel, M. Fussenegger, Curr. Opin. Chem. Biol. 2015, 28, 29-38; e) M.
Xie, V. Haellman, M. Fussenegger, Curr. Opin. Biotechnol. 2016, 40,
139-148.
Scheme 4. Schematic representation of time-dependent reduction of
enrofloxacin 1 by ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R programmed by
esterase activation and urease inhibition a). Time dependent reduction of
enrofloxacin 1 in the presence of ATHase, urease and esterase (solid line),
ATHase and esterase (dashed line) and ATHase and urease (dotted line) b).
Experimental conditions : 25 µM ATHase [(Biot-Cp*)Ir(N^N)] 3 · Sav K121R,
1 mM enrofloxacin 2, 0.2 mg/ml urease, 150 mM Urea, 0.5 mg/ml esterase, 80
mM ethyl butyrate and 2 M HCOONa in 10 mM CHES (pHinitial = 9.0) at 37 ºC.
[5]
[6]
a) A. Zhabotinsky, Biofizika 1964, 9, 306-311. b) W. Jahnke, A. T.
Winfree, J. Chem. Educ., 1991, 68, 320-324.
a) G. M. Whitesides, R. F. Ismagilov, Science 1999, 284, 89-92; b) J. M.
Lehn, Angew. Chem., Int. Ed. 2015, 54, 3276-3289; Angew. Chem.
2015, 127, 3326-3340
[7]
[8]
a) L. Heinen, A. Walther, Soft Matter 2015, 11, 7857-7866; b) B. A.
Grzybowski, W. T. S. Huck, Nat. Nanotechnol. 2016, 11, 584-591.
a) S. N. Semenov, A. S. Wong, R. M. van der Made, S. G. Postma, J.
Groen, H. W. van Roekel, T. F. de Greef, W. T. Huck, Nat. Chem. 2015,
7, 160-165; b) A. S. Wong, S. G. Postma, I. N. Vialshin, S. N. Semenov,
W. T. Huck, J. Am. Chem. Soc. 2015, 137, 12415-12420; c) S. N.
Semenov, L. J. Kraft, A. Ainla, M. Zhao, M. Baghbanzadeh, V. E.
Campbell, K. Kang, J. M. Fox, G. M. Whitesides, Nature 2016, 537,
656-660; d) J. J. t. Armao, J. M. Lehn, J. Am. Chem. Soc. 2016, 138,
16809-16814; e) B. Yurke, A. J. Turberfield, A. P. Mills, Jr., F. C.
Simmel, J. L. Neumann, Nature 2000, 406, 605-608; f) J. Boekhoven, A.
M. Brizard, K. N. Kowlgi, G. J. Koper, R. Eelkema, J. H. van Esch,
Angew. Chem., Int. Ed. 2010, 49, 4825-4828; Angew. Chem. 2010, 122,
4935-4938; g) J. Boekhoven, W. E. Hendriksen, G. J. Koper, R.
Eelkema, J. H. van Esch, Science 2015, 349, 1075-1079; h) J.
Boekhoven, J. M. Poolman, C. Maity, F. Li, L. van der Mee, C. B.
Minkenberg, E. Mendes, J. H. van Esch, R. Eelkema, Nat. Chem. 2013,
5, 433-437; i) S. Ogi, T. Fukui, M. L. Jue, M. Takeuchi, K. Sugiyasu,
Angew. Chem., Int. Ed. 2014, 53, 14363-14367; Angew. Chem. 2014,
126, 14591-14595; j) T. Heuser, A. K. Steppert, C. M. Lopez, B. Zhu, A.
Walther, Nano Lett. 2015, 15, 2213-2219; k) T. Heuser, E. Weyandt, A.
Walther, Angew. Chem., Int. Ed. 2015, 54, 13258-13262; Angew. Chem.
In summary, we have demonstrated the temporal control of an
ATHase upon combining it in-parallel with an enzyme that slowly
releases an inhibitor as a response to an external stimulus. The
reaction network is maintained off-equilibrium by addition of acid
which acts as an activator to fuel the reaction. Upon stalling,
neutralization of the inhibitor by addition of a fast activator allows
to restore enzymatic activity. Accordingly, the operational time-
window of the ATHase can be programmed at will. This self-
regulating, off-equilibrium feature is orchestrated by a feedback
mechanism reminiscent of complex cellular networks, Scheme
1c. We anticipate that the concept of temporal catalyst activation
will contribute to engineer complex, bottom-up (bio)chemical
reaction networks that combine both natural and artificial
enzymes. In particular, the prospect of exploiting a pH switch,
may allow to specifically turn on the ATHase activity in cancer
cells where the pH is lower than in healthy cells.
This article is protected by copyright. All rights reserved.

10.1002/anie.201702181
Angewandte Chemie International Edition
COMMUNICATION
2015, 127, 13456-13460; l) E. Jee, T. Bansagi, A. F. Taylor, J. A.
Pojman, Angew. Chem., Int. Ed. 2016, 55, 2127-2131; Angew. Chem.
2016, 128, 2167-2171; m) S. G. Postma, I. N. Vialshin, C. Y. Gerritsen,
M. Bao, W. T. Huck, Angew. Chem., Int. Ed. 2017, 56, 1794-1798;
Angew. Chem. 2017, 129, 1820-1824.
F. Hollmann, T. R. Ward, ACS Catal. 2016, 6, 3553-3557; c) Y.
Okamoto, V. Kohler, T. R. Ward, J. Am. Chem. Soc. 2016, 138, 5781-
5784.
[11] a) M. Jeschek, R. Reuter, T. Heinisch, C. Trindler, J. Klehr, S. Panke, T.
R. Ward, Nature 2016, 537, 661-665; b) H. M. Key, P. Dydio, D. S.
Clark, J. F. Hartwig, Nature 2016, 534, 534-537.
[9]
a) M. E. Wilson, G. M. Whitesides, J. Am. Chem. Soc. 1978, 100, 306-
307; b) T. Heinisch, T. R. Ward, Acc. Chem. Res. 2016, 49, 1711-1721;
c) F. Yu, V. M. Cangelosi, M. L. Zastrow, M. Tegoni, J. S. Plegaria, A.
G. Tebo, C. S. Mocny, L. Ruckthong, H. Qayyum, V. L. Pecoraro,
Chem. Rev. 2014, 114, 3495-3578; d) T. Ueno, S. Abe, N. Yokoi, Y.
Watanabe, Coord. Chem. Rev. 2007, 251, 2717-2731; e) T. Hayashi, Y.
Sano, A. Onoda, Isr. J. Chem. 2015, 55, 76-84; f) J. C. Lewis, Curr.
Opin. Chem. Biol. 2015, 25, 27-35; g) P. J. Deuss, R. den Heeten, W.
Laan, P. C. Kamer, Chem. Eur. J. 2011, 17, 4680-4698; h) O. Pamies,
M. Dieguez, J. E. Backvall, Adv. Synth. Catal. 2015, 357, 1567-1586; i)
J. Bos, G. Roelfes, Curr. Opin. Chem. Biol. 2014, 19, 135-143.
[12] T. Heinisch, K. Langowska, P. Tanner, J. L. Reymond, W. Meier, C.
Palivan, T. R. Ward, ChemCatChem 2013, 5, 720-723.
[13] a) I. Turel, Coord. Chem. Rev. 2002, 232, 27-47; b) S. T. Nguyen, X. Y.
Ding, M. M. Butler, T. F. Tashjian, N. P. Peet, T. L. Bowlin, Bioorg. Med.
Chem. Lett. 2011, 21, 5961-5963.
[14] a) E. Katz, V. Privman, Chem. Soc. Rev. 2010, 39, 1835-1857; b) M.
Motornov, J. Zhou, M. Pita, V. Gopishetty, I. Tokarev, E. Katz, S. Minko,
Nano Lett. 2008, 8, 2993-2997; c) I. Tokarev, V. Gopishetty, J. Zhou, M.
Pita, M. Motornov, E. Katz, S. Minko, ACS Appl. Mater. Interfaces 2009,
1, 532-536.
[10] a) V. Kohler, Y. M. Wilson, M. Durrenberger, D. Ghislieri, E. Churakova,
T. Quinto, L. Knorr, D. Haussinger, F. Hollmann, N. J. Turner, T. R.
Ward, Nat. Chem. 2013, 5, 93-99; b) Y. Okamoto, V. Kohler, C. E. Paul,
This article is protected by copyright. All rights reserved.

10.1002/anie.201702181
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Cross-regulation of an artificial
metalloenzyme is enabled upon
combining it with a natural enzyme.
The enzyme urease produces
ammonia which acts as reversible
inhibitor of the artificial transfer
hydrogenase. Addition of acid, which
acts as fuel, maintains the system
out-of-equilibrium until it is
Yasunori Okamoto, Thomas R. Ward*
Page No. – Page No.
Cross-Regulation of an Artificial
Metalloenzyme
completely consumed.
This article is protected by copyright. All rights reserved.