Bioorthogonal Enzymatic Activation of Caged Compounds

Article abstract of DOI:10.1002/anie.201506739

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences.

Full text of DOI:10.1002/anie.201506739

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International Edition: DOI: 10.1002/anie.201506739
German Edition: DOI: 10.1002/ange.201506739
Protein Engineering
Hot Paper
Bioorthogonal Enzymatic Activation of Caged Compounds
Cornelia Ritter, Nathalie Nett, Carlos G. Acevedo-Rocha, Richard Lonsdale, Katja Kräling,
Felix Dempwolff, Sabrina Hoebenreich, Peter L. Graumann, Manfred T. Reetz,* and
Eric Meggers*
Abstract: Engineered cytochrome P450 monooxygenase var-
iants are reported as highly active and selective catalysts for the
bioorthogonal uncaging of propargylic and benzylic ether
protected substrates, including uncaging in living E. coli.
observed selectivity is supported by induced-fit docking and
molecular dynamics simulations. This proof-of-principle study
points towards the utility of bioorthogonal enzyme/protecting
group pairs for applications in the life sciences.
B
ioorthogonal chemistry allows the execution of chemical
reactions inside living cells without interfering with native
biochemical processes.[1] Originally mainly focusing on stoi-
chiometric and uncatalyzed reactions, recent efforts aim at
identifying catalysts to selectively recognize specific func-
tional groups uncommon in nature and facilitate their trans-
formation within a complex biological system.[2–6] Despite the
advantages of such systems, balancing high reactivity at low
substrate concentrations and inertness towards countless
cellular molecules is a formidable challenge for the design
of small-molecule catalysts, and reported examples of artifi-
cial bioorthogonal catalysts with high turnovers are rare.[2]
The undeniable advantage of signal amplification through
catalytic turnover has been successfully exploited in the area
of enzyme-based bioimaging and sensing.[7] Enzymes have
evolved over millions of years to be efficient biocatalysts,
working optimally and selectively in diverse biological
environments. Furthermore, protein engineering provides
powerful tools such as directed evolution[8] to improve
Figure 1. The concept of bioorthogonal enzymatic deprotection of
caged compounds realized through the P450-catalyzed oxidative cleav-
age of propargylic and benzylic ethers.
enzyme properties, including thermal or chemical stability,
enhanced activity, and stereo- and regioselectivity towards
native and non-native substrates. We thus reasoned that
engineered enzymes could expand the toolbox of bioorthog-
onal chemistry by selectively catalyzing the cleavage of an
artificial protecting group in living cells (Figure 1). Such
protecting group/enzyme pairs could address the performance
issues observed in chemical- or light-induced uncaging
reactions.[2,9]
Initially, suitable enzyme/protecting group pairs were
identified, ideally featuring bioorthogonality, which means
that the protecting group is completely stable in a biological
environment and is cleaved efficiently and selectively solely
by the engineered enzyme of choice. Based on these criteria,
we chose non-native propargylic ethers as alcohol-protecting
groups to be cleaved by a member of cytochrome P450
monooxygenase (CYP) family (see Scheme S1 in the Sup-
porting Information for mechanistic details). We hypothe-
sized that propargylic ethers would not be recognized as
substrates by natural enzymes but only by engineered CYPs.
As a model system, we used P450BM3 (CYP102A1), a soluble
fatty acid hydroxylase from Bacillus megaterium,[10] which is
a highly active, selective, and versatile biocatalyst that has
been demonstrated to be tunable for many different applica-
tions.[11,12] As a fusion protein carrying its own reductase
domain, P450BM3 is self-sufficient and depends only on oxygen
and its cofactor NADPH. As model reactions, we used ether-
protected fluorophores 1–3, which are nonfluorescent but
recover their fluorescence upon deprotection, so that ether
cleavage could be detected in a straightforward fashion
through fluorescence measurements. Based on known meth-
ods[13,14] we screened cytochrome P450BM3 libraries compris-
[*] C. Ritter, N. Nett, Dr. C. G. Acevedo-Rocha, Dr. R. Lonsdale,
K. Kräling, Dr. S. Hoebenreich, Prof. Dr. P. L. Graumann,
Prof. Dr. M. T. Reetz, Prof. Dr. E. Meggers
Fachbereich Chemie, Philipps-Universität Marburg
Hans-Meerwein-Straße 4, 35043 Marburg (Germany)
E-mail: meggers@chemie.uni-marburg.de
Dr. C. G. Acevedo-Rocha, Dr. R. Lonsdale, Prof. Dr. M. T. Reetz
Max-Planck-Institut für Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr (Germany)
E-mail: reetz@mpi-muelheim.mpg.de
Dr. C. G. Acevedo-Rocha, Dr. F. Dempwolff, Prof. Dr. P. L. Graumann
LOEWE Zentrum für Synthetische Mikrobiologie (SYNMIKRO)
Hans-Meerwein-Straße, 35043 Marburg (Germany)
Dr. C. G. Acevedo-Rocha
Max-Planck-Institut für terrestrische Mikrobiologie
Karl-von-Frisch-Straße 10, 35043 Marburg (Germany)
Prof. Dr. E. Meggers
College of Chemistry and Chemical Engineering, Xiamen University
Xiamen 361005 (P. R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201506739.
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ing more than 1000 enzyme variants capable of oxidizing
hydrophobic compounds (see the Supporting Information).
First, a bis-propargylic ether derivative of fluorescein
(1a), together with the related bis-allyl- (1b) and bis-benzyl-
protected derivatives (1c; Figure 2) was screened for fluores-
cence development induced by the P450BM3 variants. As
shown in Figure 3, only a few enzyme variants were found to
convert the allyl derivative 1b, while no mutants were found
to cleave the benzyl diether 1c. However, a number of
enzyme variants displayed remarkably high activity for
cleavage of the propargylic diether 1a, the most prominent
example being mutant TFFIS (mutations R47T/S72F/A82F/
F87I/L437S). Importantly, mutant TFFIS preferentially acti-
vates the propargylic diether 1a over 1b (weak activation) or
1c (no activation), while wild type (wt) P450BM3 does not
activate 1a, thus demonstrating a compelling degree of
selectivity. HPLC analysis revealed the mono-deprotection
of 1a (see Figure S1 in the Supporting Information). The
distinct suitability of the propargylic protecting group was
confirmed by experiments with the alkyne derivatives 1d–f.
Whereas a large number of P450BM3 mutants display weak
activity towards the 2-butynyl diether 1d, no active variants
could be found for the alkynes 1e and 1 f (Figures 2 and 3).
Encouraged by these results, the propargylic ether deriv-
atives (2a and 3a) of two coumarins (2 and 3) were
investigated and the respective 2-butynyl (2d and 3d) and
benzyl (2c and 3c) ethers were used as references. 4-
Methylumbelliferone (2) is an established drug used in bile
therapy[15] and known for its activity as an anticancer agent
that is effective against various cancer types.[16] Its ethers are
closely related to a known probe for CYP levels in clinical
samples.[17] The second compound, 3-carboxycoumarin (3), is
a known substrate for similar reactions catalyzed by P450
variants.[18] Upon screening, mutant M01 (R47L/F87V/
L188Q/E267V/G415S)[19] was capable of activating the prop-
argylic ethers 2a and 3a, whereas mutant WMV (R47W/
A82M/F82V) displayed very high activity towards the ben-
zylic ethers 2c and 3c. These results are particularly note-
worthy since alkynes are known to act as suicide inhibitors,
irreversibly inactivating CYPs through alkylation of the heme
group.[20]
The most prominent variants were purified and charac-
terized biochemically by using defined enzyme concentra-
tions and the same reaction conditions used for the screening
procedure (see the Supporting Information). It was thus
possible to cancel out differences in expression levels present
in lysates, thereby allowing the comparison of our most
promising hits (Figure 4). In the case of substrate 1a, the most
active mutant TFFIS yields a 52-fold higher fluorescence
response than the wt, corresponding to a turnover number
(TON) of 425. The Michaelis–Menten parameters KM and kcat
were determined to be 3.7 mm and 0.26 sÀ1, respectively (kcat
/
KM = 70.3 mmÀ1 sÀ1). None of the other hits was able to
convert any of the fluorescein substrates. While the highly
active mutant WMV (for 3d: TON > 5000, KM = 7.2 mm, kcat
=
550 sÀ1; kcat/KM = 7.6 ” 104 mmÀ1 sÀ1) activated all of the cou-
marin-derived substrates (2a, 2c, 3c, and 3d), the more
selective mutant M01 showed activity towards substrates 2a
and 2c only, at about 4-fold higher activity than the wt
(TON = 47). This means that even compounds with very
similar core structures can be differentiated by various
mutants, but this gain in selectivity comes with an activity
penalty, for example, WMV shows 16-fold higher activity than
M01 towards substrate 3d. Previous reports for 3c[18c] with
other P450BM3 mutants achieved only a 7-fold increase at
similar concentrations in a different buffer[21] after one round
of random directed evolution, which differs from our library
Figure 2. Model compounds screened. The first- and second-genera-
tion compounds are fluorescein derivatives, whereas the third-gener-
ation compounds are based on coumarins.
Figure 3. Activity of the P450BM3 mutants towards different caged
compounds arranged in a 96-well microtiter plate (MTP) format.
Fluoresceins 1a–f (green) and coumarin-based substrates 2a,c,d (red)
and 3a,c,d, (blue) were used for library screening. Fluorescence
intensities are normalized to the lowest/highest values included in the
plates, with brighter colors showing higher activity (see the Supporting
Information for original values).
Figure 4. In vitro characterization. Enzymatic activity of P450BM3 var-
iants towards caged compounds at fixed enzyme concentrations
(generally 100 nm), normalized to the largest observed fluorescence
response for each substrate (see Appendix 2 in the Supporting
Information for original data).
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Figure 5. Modeling results. Docking poses for a) substrate 1a in the
TFFIS mutant, and b) substrate 3d in the WMV mutant. Distances
r(O–H) between the substrate and active center are 3.64 Š and 2.29 Š,
respectively. Highlighted are the oxidized prosthetic heme groups,
substrate, and mutated residues.
Figure 6. Live-cell experiments. E. coli expressing TFFIS (panels a–c)
show a high fluorescence signal, thus indicating deprotection of
substrate 1a. No significant signal level is observed in the wt (panels
d–f). Shown are the fluorescence channel (left), the optical channel
(middle), and an overlay of the two channels (right). Scale bars: 2 mm.
mutants where the active site was targeted according to the
recommended guidelines.[22]
To provide structural insight into binding modes and
substrate selectivity of the most active substrate/mutant
combinations, docking and molecular dynamics (MD) simu-
lations were performed (Figure 5 and the Supporting Infor-
mation). Briefly, substrates 1a and 3d were modeled in the
TFFIS and WMV mutants, respectively. Since there are no
published crystal structures of the mutants studied here,
mutations were computationally modeled in the wt crystal
structure (PDB code 1BU7).[23] In both cases, stable binding
modes of the substrates were obtained.
ethers. We also explain the basis of the selectivity of the most
promising mutants by using docking and molecular dynamics
simulations. Finally, we show that these P450 mutants
successfully catalyze the uncaging reaction in living E. coli.
Such bioorthogonal engineered enzyme/protecting group
pairs may find applications in the life sciences, for instance,
for the release of imaging agents or the catalytic activation of
prodrugs at their site of action.
The computational results exhibit substrate binding
orientations in the active site of the corresponding variants
that enable hydroxylation of the protecting group by the
oxidizing iron–oxo species (Compound I). Binding of 1a in
the TFFIS mutant (Figure 5a) involves a p-stacking inter-
action with the S72F side chain, which positions the protecting
group for transformation. On the other hand, substrate 3d can
enter deep into the pocket of the WMV mutant, thereby
placing the methylene hydrogen atom close to the heme
group and allowing fast turnover, in accordance with the
kinetic profile (see Table S3). All of the found hits have
a mutation at position R47, which has been proposed to play
a crucial role in substrate binding.[24] Since this residue is the
only charged residue around the binding pocket, substitution
for a polar but neutral or even aliphatic amino acid, as in this
case, has a large effect on catalytic efficiency.
The ability of the mutants to deprotect the model
compounds in living E. coli was explored (Figure 6). High
fluorescence intensity within the cells and low efflux were
observed for 1a, as can be seen from supernatant and lysate
analyses (Figure S3). Contrary to expectations based on
previous results by Ruff et al.,[18c] substrates 3c and 3d
could not be monitored in this way. High efflux of 3 raised the
background to a level too high for imaging, even after
repeated washing of the cells. Thus, unlike fluorescein
derivative 1a, the coumarin-based substrates were not
suitable for imaging, but nevertheless the reaction in living
cells was successful in all tested cases.
Acknowledgements
This work was supported by a LOEWE Research Cluster of
the State of Hesse (SynChemBio), the University of Marburg
and the Max-Planck Society.
Keywords: bioorthogonal chemistry · directed evolution ·
cytochromes · protecting groups · protein engineering
How to cite: Angew. Chem. Int. Ed. 2015, 54, 13440–13443
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In summary, we report the identification of engineered
CYP variants that are able to efficiently and selectively
uncage compounds protected with propargylic and benzylic
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Received: July 21, 2015
Revised: August 14, 2015
Published online: September 10, 2015
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