Optical control of enzyme enantioselectivity in solid phase

Article abstract of DOI:10.1021/cs401115s

A lipase was immobilized on transparent agarose microspheres and genetically engineered to specifically anchor photochromic molecules into its catalytic site. Several combinations of azobenzene and spiropyran groups were conjugated to cysteines introduced at different positions near the active center. Light modulated the catalytic properties of the resulting solid bioconjugates, and such modulation depended on both the nature of the photochromic compound and the anchoring position. Covalent anchoring of azobenzene derivatives to the residue 295 of the lipase 2 from Bacillus thermocathenolatus triggered lipase preference for the S isomer under UV light, whereas visible light promoted preference for the R isomer. Molecular dynamics simulations indicate that conjugating photochromic compounds into the catalytic cavity allows manipulating the steric hindrance and binding energy of the substrates, leading to an enantioselective molecular fit in certain cases. Using this approach, we report for the first time the control of enzyme properties using light in the solid phase.

Full text of DOI:10.1021/cs401115s

Research Article
pubs.acs.org/acscatalysis
Optical Control of Enzyme Enantioselectivity in Solid Phase
Antoni Bautista-Barrufet,■,†,¶ Fernando Lopez-Gallego,*,■,‡ Víctor Rojas-Cervellera,§ Carme Rovira,§,⊥
́
Miquel A. Pericas,¶ Jose M. Guisan,‡ and Pau Gorostiza*,†,⊥,□
̀
́
́
†Institut de bioenginyeria de Catalunya (IBEC), C/Baldiri Reixac 15-21, Barcelona 08028, Spain
‡Instituto de Catal
isis y Petroleoquímica (ICP-CSIC), C/Marie Curie n 2°, Madrid 28029, Spain
́
§
́
́
Departament de Quımica Organ
⊥Institucio
Catalana de Recerca i Estudis Avanca
¶Institut Catala
d’Investigacio Química (ICIQ). Avinguda Països Catalans 16, Tarragona 43007, Spain
□Centro de Investigacion
Biomedica en Red sobre Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/Poeta Mariano
̀
ica, Universitat de Barcelona (UB), Mart ı i Franques
̀
1, Barcelona 08028, Spain
́
̧
ts (ICREA), C/Lluís Companys 23, Barcelona 08010, Spain
̀
́
́
́
Esquillor s/n, Zaragoza 50018, Spain
S
* Supporting Information
ABSTRACT: A lipase was immobilized on transparent
agarose microspheres and genetically engineered to specifically
anchor photochromic molecules into its catalytic site. Several
combinations of azobenzene and spiropyran groups were
conjugated to cysteines introduced at different positions near
the active center. Light modulated the catalytic properties of
the resulting solid bioconjugates, and such modulation
depended on both the nature of the photochromic compound
and the anchoring position. Covalent anchoring of azobenzene
derivatives to the residue 295 of the lipase 2 from Bacillus thermocathenolatus triggered lipase preference for the S isomer under
UV light, whereas visible light promoted preference for the R isomer. Molecular dynamics simulations indicate that conjugating
photochromic compounds into the catalytic cavity allows manipulating the steric hindrance and binding energy of the substrates,
leading to an enantioselective molecular fit in certain cases. Using this approach, we report for the first time the control of
enzyme properties using light in the solid phase.
KEYWORDS: lipase, azo compounds, photochromism, chemical modification, immobilization
amphiphilic domain (named as “lid”) that triggers the catalytic
mechanism in the presence of hydrophobic substrates.5 This
class of enzymes is the paradigm of enantioselective
biocatalysis, and their hydrolytic rate and enantiomeric excess
can be enhanced by adjusting the position of the substrate into
the hydrophobic cavity.6 A plethora of methodologies to alter
the lipase catalytic mechanism have been devised, including
enzyme engineering, reaction media engineering, immobiliza-
tion, and chemical modification.6−8 We have recently reported
the alteration of both activity and selectivity of lipase 2 from
Bacillus thermocathenolatus (BTL2) using site-directed chemical
modification in the solid phase.9 Building upon this method-
ology, we pursued photocontrol of BTL2 catalytic properties by
tethering a photochromic group inside the enzyme active
center.
INTRODUCTION
■
Isolated enzymes can catalyze organic transformations at both
high yield and high enantioselectivity under mild conditions.
However, in situ modulation of catalytic properties has proven
a great challenge, as indicated by the lack of methods to
regulate enzyme enantioselectivity during in vitro biotransfor-
mations. The dynamic control of enzyme selectivity for in vitro
reactions schemes is thus an unmet need, especially in the
context of cascade reactions catalyzed by multienzyme systems,
in which biocatalysts must be switched on and off in situ
according to system requirements. Optical control1 offers the
possibility to remotely manipulate enzyme activity using
spatiotemporally designated patterns of illumination.2 More-
over, the immobilization of these engineered biocatalysts would
enable their reuse as well as their incorporation into
nanodevices.3 Here, we present a rational approach to
conjugate photochromic compounds to an immobilized
enzyme in a site-directed manner and demonstrate for the
first time the regulation of its enantioselectivity with light.
Lipases are serin−threonin hydrolases that naturally catalyze
the hydrolysis of lipids and are widely applied in chemical
processes from research laboratories to industrial plants.4 Most
lipases present a hydrophobic active site shielded by an
RESULTS AND DISCUSSION
■
Site-Directed Chemical Modification of BTL2 in Solid
Phase. We chemically modified BTL2 with different photo-
Received: November 25, 2013
Revised: February 1, 2014
Published: February 13, 2014
© 2014 American Chemical Society
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chromic molecules. As a result of the reversible transformation
of the photochromic group anchored to the protein scaffold, we
expected the active center conformation to change under
different illumination conditions (Figure 1). To test whether
Figure 1. Site-directed chemical modification of BTL2 with
phoswitchable molecules. Reversible conformational changes of
semisynthetic binding pocket induced by light. BTL2 immobilized
on agarose beads activated with cyanogen bromide groups has been
site-directed-modified with photoswitchable molecules. Since the
photosensitive molecules have been closely anchored to the catalytic
residues, enzyme properties can be modulated.
those changes availed in altering the enzyme activity, we
evaluated several positions located at the substrate binding
pocket and two compounds displaying different photochromic
processes (Figure 2A,B). In particular, an iodoacetate−
spiropyran (1, Figure 2A) and an azobenzene−methylthiosul-
fonate (2, Figure 2B) were specifically conjugated to a unique
cysteine at positions 17, 187, 245, 295, and 320 of BTL2. These
positions were rationally chosen because they are important
either for the substrate binding (17, 245 and 320) or for the lid
aperture during the catalysis (187 and 295) (Supporting
Information (SI) Figure S1 and Table S1).5 The unique
cysteine at the specific position was introduced by site-directed
mutagenesis using a cysteine-less BTL2 variant that was created
in previous work9 to avoid unspecific chemical modification.
Both catalytic rate and enantioselectivity were assayed toward
a survey of esters, under different light conditions (see the SI
for details on the synthesis, mutagenesis, purification and site-
selective chemical modification in solid phase of the enzymes).
The conjugation took place in the solid phase by incubating the
immobilized BTL2 in a photochromic compound solution. The
yield of the chemical modification was evaluated by thiol
titration. As an example, 94 9 (%) of the BTL2 active centers
were modified with molecule 1 at position 245. The
conjugation procedure was simplified by solid-phase prepara-
tion because excess reagents and solvents could be easily
removed by vacuum filtration. The site-directed chemical
modification was confirmed by mass spectrometry (SI Figure
S2)
Spectroscopic Characterization of the Photosensitive
Biocatalysts. The photochromic biocatalysts were spectro-
scopically characterized under different illumination conditions.
Ultraviolet (UV) light transforms the apolar spiropyran 1 into
the planar, polar merocyanine form (Figure 2A) and causes the
isomerization of azo derivative 2 from the extended trans
configuration to the bent cis form (Figure 2B). In both cases,
the initial state is reversibly recovered under visible illumination
or after thermal relaxation in darkness (Figure 2C,D). It is
noteworthy that photochromic molecules tethered to immobi-
lized BTL2 (1b and 2b) presented absorbance spectra similar
to those of the molecules in solution. UV light increased the
absorbance of compound 1 at 550 nm, as reported10 (Figure
2C, inset) and decreased the absorbance compound 2 at 350
nm, as described for similar azobenzenes11 (Figure 2D, inset).
Figure 2. Photochromic properties of different immobilized
bioconjugates. Photoswitchable equilibrium undergone by spiropyran
(A) and azobenzene (B) molecules. The spiropyran compound is
activated by an iodoacetate reactive group (1a) to specifically react to
cysteines on the protein structure, resulting in covalent and irreversibly
anchored (1b) bioconjugates. In the same way, the azobenzene
compound was activated with methylthiosulfonate groups (MTS) (2a)
that selectively react to cysteine, as well, but form covalent and
reversible bioconjugates (2b). Spectroscopic characterization of
immobilized BTL2 bioconjugates modified with either spiropyran
(C) or azobenzene (D) molecules (see Supporting Information). Inset
figure depicts the absorbance spectrum of solid bioconjugates
illuminated with either visible (green line) or UV (violet line) light.
The arrows point out the effect of the light switch on the absorbance
maximum of the different molecules. The main graph represents the
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Figure 2. continued
evolution of active site states under different light conditions. Red
circles correspond to wild type and, consequently, nonmodified BTL2;
black squares, to BTL2-modified at position 245. After illuminating
solid photochromic bioconjugates with UV lights, the relaxation of the
excited compound was monitored along the time in darkness.
Afterward, the solid preparations were subjected to light cycles to
study the photoisomerization of the anchored molecules into the
active site environment.
However, the half-life of thermal relaxation and the stationary
state achieved in the dark by both photochromic molecules
were substantially altered by coupling to the protein.12 For
example, after UV illumination, the soluble 1a relaxation rate
under darkness was 20% faster than the same molecule
anchored to position 245 in the BTL2 active site (1b). In fact,
80% of soluble 1a was relaxed after 24 h in darkness, but only
60% relaxation was observed after 24 h for 1a conjugated to the
protein scaffold (1b). Moreover, 1a and 1b preparations only
partially relaxed in the dark and required visible illumination to
completely convert them back to the spiropyran form (SI
Figure S3A, Figure 2C). Therefore, the alteration of the
“reversible photo-equilibrium” suggests that the protein
environment could stabilize the polar merocyanine, as
previously reported.13
Effect of Light on Immobilized BTL2 Catalytic Proper-
ties. Catalytic reactions with several conjugated BTL2 variants
were carried out under different illumination conditions, and
the activities were measured using colorimetric assays (see the
SI). Figure 3A and B shows the hydrolytic activity of different
immobilized bioconjugates under UV and visible light. In
general, conjugation of photochromic molecules led to a
reduction in lipase activity, but several variants displayed a
catalytic rate that was moderately light-dependent, thus
validating the strategy to photocontrol an enzyme with a
shielded active site. As an example, BTL2 conjugated with 1 at
positions 187 and 245 (BTL2−187C-1 and BTL2−245C-1)
and with 2 at position 245 (BTL2−245C-2) presented a
relative 1.5-fold higher activity under UV light than under
visible light (Figure 3A and B). These results demonstrate that
the anchoring site of the photochromic group in the lipase is
critical to control its hydrolytic activity with light and constitute
the first demonstration of enzyme photoregulation in the solid
state using porous materials as carriers. The choice of agarose
beads as carriers was key because its transparency allows light
passing through the pores and illuminating the enzymes
attached to the solid structure, thereby activating the
photochromic molecules anchored to the protein.
These novel results prompted us to examine enzyme
enantioselectivity, arguably one of the most outstanding
properties of lipases.6 To that end, we tested racemic mixtures
as substrates and measured the chirality of products yielded by
different BTL2 bioconjugates under UV and visible illumina-
tion. The enantioselectivity of modified BTL2 variants toward
two different racemic esters, 2-butyryloxy-2-phenylacetic acid
(rac-7) and 1-phenyl-ethyl acetate (rac-8), is shown in Figure
3C and D, respectively. In the first case, site-directed
modification of BTL2 with compound 1 tended to preferen-
tially hydrolyze the S enantiomer, inverting the natural
preference by the R enantiomer displayed by the native
enzyme. In contrast, modification with compound 2 enhanced
the enantiomeric excess values of the native enzyme (see
Figure 3. Analysis of the enzyme properties of photoswitchable lipase
under different illumination conditions. Esterase activity of different
immobilized variants of BTL2 modified with either 1a (A) or 2a (B)
compounds under light at different wavelengths. Hydrolytic activity of
the different chemically modified BTL2 variants were measured under
either visible light (green bars) (500 and 460 nm for 1b and 2b
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controlled transesterification reaction using glycerol (3) and
methyl butyrate (4) as substrates (Figure 4). It has been
Figure 3. continued
bioconjugates respectively) or UV light (violet bar) (380 and 365 nm
for 1b and 2b bioconjugates, respectively). The spherical points show
the photoisomerization ratio defined as the coefficient between the
specific enzyme activity under UV and visible lights. Values close to 1
mean a non photoswitchable lipase, and ratios higher or lower than 1
mean photoswitchable lipases. Selective hydrolysis of 2-butyryloxy-2-
phenylacetic acid (rac-7) (C) and 1-phenyl-ethyl acetate (rac-8) (D)
to their corresponding chiral alcohols 3-phenyl-2-hydroxypropionic
acid (9) and 1-phenyl ethanol (10) catalyzed by different BTL2
bioconjugates modified with either 1a or 2a compounds. Effect of light
on the lipase selectivity was determined by measuring the
enantiomeric excess (ee; %) of the different bioconjugates modified
at different positions under either visible (green bars) or UV (violet
bars) light. For bioconjugates 1b, visible and UV wavelengths were 500
and 380 nm; for bioconjugates 2b, they were 460 and 365 nm for
visible and UV light, respectively.
complete results in SI Tables S2 and S3). Photocontrol of the
enantioselectivity depended both on the conjugation site and
on the nature of the photochromic molecule, unlike activity
that depended on only the conjugation position. In this regard,
the enantioselectivity for the hydrolysis of rac-7 could be
inverted with light by using BTL2−320C-1 (the S enantiomer
is favored under visible light) and BTL2−295C-2 (the S
enantiomer is favored under UV light) (Figures 3C and SI S4).
On the other hand, enantioselectivity changes were also
observed in the hydrolysis of rac-8 catalyzed by BTL2−
187C-1 and BTL2−320C-1 (the R enantiomer is favored under
visible light) (Figure 3D). In particular, the enantioselectivity of
BTL2−187C-1 toward rac-8 under visible light was 3 times
higher than under UV light and approached that of the native
enzyme.
Position 320 located at the binding pocket and the contact
region between the lid domain and the active site are “hot-
spots” to anchor photochromic molecules that effectively
modulate the enzyme enantioselectivity by the action of the
light. Similarly, chemical modification of position 320 of BTL2
with alkanes modified both the activity and the enantiose-
lectivity of the immobilized BTL2.9
In principle, this approach can be expanded to other
enantioselective enzymes. In fact, the activity of several
enzymes can be modulated by light regardless both the
morphology of their active sites and the catalytic mechanisms
(endonucleases, carbonic anhydrase, etc.).14−16 Therefore,
these light-driven changes in enzyme enantioselectivity
constitute a breakthrough in the regulation of biocatalysis for
in vitro applications that complements engineered17 and
artificial18−20 enzyme strategies to provide reversibility and
remote control and largely outperforms previous attempts using
additives in the reaction media.9 Moreover, this work proves for
the first time that light can also modulate enzyme properties in
the solid phase as well as it does in solution. The resulting
photochromic immobilized biocatalysts might be used in a fix-
bed reactor for a continuous process that is able to respond to
external signals, such as the light. This opens new opportunities
for the regulation of biocatalytic systems formed by isolated
enzymes.
Figure 4. Kinetically controlled synthesis of 2-O-butyryl-glycerol (5)
catalyzed by BTL2 modified with 1a at position 245 . The synthesis of
5 was carried using glycerol (3) and methyl butyrate (4) as substrates.
In this reaction, the enzyme can also catalyze the hydrolytic reaction of
the substrate 4, yielding butyric acid (6) and methanol as the products.
The light was switched every 30 min during the reaction, and the
substrates and products were analyzed by HPLC to study the
simultaneous hydrolytic and synthetic reactions of the mentioned
bioconjugate. Synthetic (blue bars) and hydrolytic (red bars) rates
were determined under different light conditions. Error bars show the
standard deviation of two independent measurements subjected to t
test analysis, where (*)p < 0.05, (**)p < 0.01 are the significant
differences between hydrolytic and synthetic rates and n.s. means no
significant differences between those rates. The black circles and solid
black line represent the ratio between the synthetic and the hydrolytic
activity of the photoswitchable bioconjugate, and the white circles and
dashed black line show the same ratio for the unmodified wild type
enzyme.
reported that BTL2 catalyzes the transesterification reaction,
yielding the corresponding glyceryl butyrate (5). However, the
enzyme can also hydrolyze 4, yielding butyric acid (6),
undesirably lowering the synthetic product yield.21 This
reaction is highly interesting from a mechanistic point of view
because it reveals that the lipase can act as synthetase and
hydrolase in the same reaction. The ratio between synthesis and
hydrolysis depends on the catalytic properties of the enzyme,
and thus, it could be, in principle, regulated by light. To test this
possibility, we carried out the transesterification reaction using
immobilized BTL2−245-1 and different light cycles. Figure 4
shows how BTL2 properties were altered by light while the
unmodified native lipase was insensitive to illumination.
Although UV light promotes an active site configuration that
can hydrolyze and synthesize at similar rates, visible light drives
an enzyme that hydrolyzes 3 times better than it synthesizes. In
the light of the statistical analysis of these results, differences
between synthetic and hydrolytic ratios were much more
significant under visible light than under UV light; therefore,
the synthesis/hydrolysis ratio can be dynamically controlled by
light, enabling the temporal control of catalytic performance of
BTL2 in mechanistic terms.
Effect of Light on Catalytic Versatility of the Photo-
chromic Biocatalysts. Since photoisomerization is expected
to produce subtle changes in the catalytic site, we evaluated the
photocontrol of the catalytic versatility of these novel
biocatalysts. For that purpose, we performed a kinetically
In Silico Studies of Photosensitive Bioconjugates.
Binding Mode of Substrate to the Active Site under
Different Light Conditions. Because the success of this
approach was built by rational design but also by testing
empirically several attachment positions and reaction con-
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ditions, we turned to computer simulations of the best
photocontrolled lipase variants to identify the principles that
could help in the optimization of further design. In particular,
we asked whether the observed enantiomeric excesses for the
different bioconjugates can be explained by the influence of
photoisomerization on the spatial availability within the active
site by altering the substrate-binding properties (energy and
conformation) or by conformational changes in the enzyme
structure. To this aim, molecular dynamics (MD) simulations
were performed on the lipase modified with either spiropyran
or azobenzene molecules (see Online Methods), followed by
separate docking calculations with the R and S enantiomers of
the 2-butyryloxy-2-phenylacetic acid (rac-7) substrate. MD
simulations have successfully been used to model conforma-
tional motions in lipases,22 including substrate binding.23 The
current MD simulations show that bioconjugation with 1 and 2
does not perturb the protein folding, and the substrate binding
pocket does not change significantly. Because the reactions
were performed under nonsaturating conditions (1 mM),
binding energy differences should reflect differences in
reactivity. Analysis of the most populated substrate docking
poses (among the ones well oriented for catalysis) (Figure 5)
residues), with the effect being more pronounced for BTL2−
295C-2 under UV-light and in the cis conformation (Figure 5
and SI Table S4). Therefore, the simulations support that the
highest enantiomeric excesses are experimentally observed for
these two bioconjugates: BTL2−320C-1(top) and BTL2−
295C-2 (bottom) under visible (left) and UV (right) light. The
differences in binding energies between the R and S
enantiomers are indicated on the figures (positive values
indicate binding that favors the S enantiomer).
Several strategies have been devised to control the catalytic
rate of enzymes with light,1 including unspecific conjugation of
photochromic compounds,13 diffusible photochromic ligands,24
and site-specific incorporation of photochromic unnatural
aminoacids.15 However, photocontrol of chirality has remained
elusive, despite the wide biochemical and biotechnological
importance of enantioselective enzymes and knowledge of their
structure and catalytic mechanisms.
Here, we have presented a rational approach to nanoengineer
immobilized enzymes by site-directed chemical modification
with photochromic compounds. We have demonstrated how
such chemical modifications enable optical regulation of BTL2
catalytic properties in different reaction schemes. The strategy
of conjugating photochromic compounds in the binding pocket
has allowed manipulation of the steric hindrance of the cavity
and achievement of an enantioselective molecular fit in certain
cases. Structural modeling and simulations provide a good basis
to understand the experimental results, pinpointing the atomic-
scale structural elements that are key to photoregulate BTL2
activity and selectivity. In particular, light acts as an external
stimulus that influences the active site spatial availability rather
than promoting a conformational change in the protein tertiary
structure (SI Figure S5).
In this way, light remotely alters the substrate binding
because the binding pocket is differently but reversibly shaped
by a photocontrolled molecular adaptor. The combination of
experiments and simulations suggests that the approach could
be extended to other structurally well-characterized enzymes or
multienzymatic complexes, providing reversible and non-
invasive modulation of different biocatalytic properties in-pot.
This novel form of enzymatic photoregulation might be useful
to externally control a branching point in cascade enzymatic
reactions. For example, in one-pot multienzymatic complexes,
including photochromic lipase, light would act as an external
stimulus, allowing directing of the chemical signals involved in
cascade chemical transformations, that is, selecting the products
to be fed as substrates into subsequent enzymatic steps. This
function is analogous to that of demultiplexer devices in
electronic circuits (SI Figure S6) and has been recently
demonstrated in organic molecular devices.25 The enzyme with
photoswitchable enantioselectivity described here thus con-
stitutes the first demonstration of a protein-based demux.26
Figure 5. Most populated docking modes of (R,S)-2-butyryloxy-2-
phenylacetic acid (rac-7) in the active site of the bioconjugates BTL2−
320C-1(top) and BTL2−295C-2 (bottom) under visible (left) and
UV (right) light. The differences in binding energies between the R
and S enantiomers are indicated on the figures (positive values indicate
binding that favors the S enantiomer).
CONCLUSION
shows that the R/S enantiomers have a similar binding energy
for the bioconjugates showing little enantiomeric excess
(BTL2−320C-1 in the merocyanine state and BTL2−295C-2
in the trans configuration, Figure 3C). In contrast, the two
enantiomers are well separated in terms of binding energy for
the bioconjugates for which the highest enantiomeric excess
was measured (BTL2−320C-1 in the spiropyran state and
BTL2−295C-2 in the cis configuration, Figure 3C). Fur-
thermore, the S enantiomer is clearly favored with respect to
the R enantiomer not only in terms of energy but also in terms
of structure (closeness of the substrate to the catalytic
■
In summary, we have presented a method to control the
catalytic properties of a lipase with light that is based on the
site-directed chemical conjugation of photochromic molecules
into the catalytic cavity. In particular, we have demonstrated for
the first time the control of enzyme enantioselectivity with light
by using chemically modified heterogeneous biocatalysts.
Molecular dynamics simulations indicated that conjugating
photochromic compounds in the active site allows manipu-
lation of the steric hindrance and binding energy of the
substrates, leading to an enantioselective molecular fit in certain
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* Supporting Information
Experimetnal details and raw data. This material is available free
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AUTHOR INFORMATION
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Corresponding Authors
*E-mail: flg802@gmail.com.
*E-mail: pau@icrea.cat.
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́
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■Antonio Bautista-Barrufet and Fernando Lopez-Gallego have
contributed equally to this work.
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n, T.; Andre, I.; Remaud-Simeon, M.
́
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Notes
The authors declare no competing financial interest.
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(26) Balzani, V.; Credi, A.; Venturi, M. Molecular Devices and
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VCH Verlag: Weinheim, 2008.
■
We are grateful for financial support from the Human Frontier
Science Program through a Career Development Award
(CDA022/2006), from the European Research Council
through the “Opticalbullet” Starting Grant (ERC-StG-
210355/2007), from the European Commission through the
“Focus” ICT-FET Grant (FP7-ICT-2009-270483), from the
Ministry of Education through Grants CTQ2008-06160 and
CTQ2011-25871, a “Juan de la Cierva” research associate
fellowship, and FPI and FPU fellowships, and from the
́
RecerCaixa and Marato de TV3 foundations.
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