Flexibility modulates the catalytic activity of a thermostable enzyme: Key information from optical spectroscopy and molecular dynamics simulation

Article abstract of DOI:10.1039/c9sm02479d

Enzymes are dynamical macromolecules and their conformation can be altered via local fluctuations of side chains, large scale loop and even domain motions which are intimately linked to their function. Herein, we have addressed the role of dynamic flexibility in the catalytic activity of a thermostable enzyme almond beta-glucosidase (BGL). Optical spectroscopy and classical molecular dynamics (MD) simulation were employed to study the thermal stability, catalytic activity and dynamical flexibility of the enzyme. An enzyme assay reveals high thermal stability and optimum catalytic activity at 333 K. Polarization-gated fluorescence anisotropy measurements employing 8-anilino-1-napthelenesulfonic acid (ANS) have indicated increasing flexibility of the enzyme with an increase in temperature. A study of the atomic 3D structure of the enzyme shows the presence of four loop regions (LRs) strategically placed over the catalytic barrel as a lid. MD simulations have indicated that the flexibility of BGL increases concurrently with temperature through different fluctuating characteristics of the enzyme's LRs. Principal Component Analysis (PCA) and the Steered Molecular Dynamics (SMD) simulation manifest the gatekeeper role of the four LRs through their dynamic fluctuations surrounding the active site which controls the catalytic activity of BGL.

Full text of DOI:10.1039/c9sm02479d

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DOI: 10.1039/C9SM02479D
Dynamical Flexibility Modulates Catalytic Activity of a Themostable Enzyme:
Key Information from Optical Spectroscopy and Molecular Dynamics
Simulation
Pritam Biswas1, Aniruddha Adhikari2, Uttam Pal3, Priya Singh2, Monojit Das4, Tanusri Saha-
Dasgupta5, Sudeshna Shyam Choudhury1*, Ranjan Das6* and Samir Kumar Pal2,4*
1Department of Microbiology,
St. Xavier’s College,
30, Mother Teresa Sarani,
Kolkata 700016, India
2Department of Chemical, Biological and Macromolecular Sciences,
S. N. Bose National Centre for Basic Sciences,
Block JD, Sector III, Salt Lake,
Kolkata 700106, India
3Technical Research Centre,
S. N. Bose National Centre for Basic Sciences,
Block JD, Sector III, Salt Lake,
Kolkata 700106, India
4Department of Zoology,
Uluberia College, University of Calcutta,
Uluberia,
Howrah 711315, India
5Department of Condensed Matter Physics and Material Sciences,
S. N. Bose National Centre for Basic Sciences,
Block JD, Sector III, Salt Lake,
Kolkata 700106, India
6Department of Chemistry,
West Bengal state University,
Barasat, Kolkata 700126, India
Corresponding authors E-mail: ranjan.das68@gmail.com (R Das)
skpal@bose.res.in (S. K. Pal)
sudeshna.s.choudhury@sxccal.edu (S. S. Choudhury)
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Abstract
Enzymes are dynamical macromolecules and their conformation can be altered via local
fluctuations of side chains, large scale loop and even domain motions which are intimately linked
to their function. Herein, we have addressed the role of dynamic flexibility in the catalytic activity
of a thermostable enzyme almond Beta-Glucosidase (BGL). Optical spectroscopy and classical
molecular dynamics (MD) simulation were employed to study thermal stability, catalytic activity
and dynamical flexibility of the enzyme. Enzyme assay reveals high thermal stability, and
optimum catalytic activity at 333K. Polarization-gated fluorescence anisotropy measurements
employing 8-Anilino-1-napthelenesulfonic acid (ANS) have indicated increasing flexibility of the
enzyme with an increase in temperature. Study of the atomic 3D structure of the enzyme shows
the presence of four loop regions (LRs) strategically placed over the catalytic barrel as a lid. MD
simulations have indicated that flexibility of BGL increases concurrently with temperature through
different fluctuating characteristics of the enzyme’s LRs. Principal Component Analysis (PCA)
and Steered Molecular Dynamics (SMD) simulation manifest the gatekeeper role of the four LRs
through their dynamic fluctuations surrounding the active site which controls catalytic activity of
BGL.
Keywords: Beta-Glucosidase; Spectroscopy; Loop Region; Molecular Dynamics simulation
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Introduction
β-Glucosidases (BGLs) constitute a subgroup of β-D-glucoside glucohydrolases (EC3.2.1.21) and
are among the most widely studied enzymes owing to their wide variety of substrates, and broad
distribution in the biological strata. BGLs are well characterized biologically important enzymes
which catalyse the transfer of glycosyl groups between oxygen nucleophiles and hydrolyze the
terminal, non-reducing β-D-glucosyl residues and thereby the terminal sugar moiety. 1-3 BGL have
attracted considerable interests due to its importance in various biotechnological processes like
hydrolysis of pharmaceutical ingredients, production of bio-ethanol from agricultural wastes. 4, 5
BGL is used extensively in the wine, tea and juice industries.6-8 In addition to their use in industry,
BGLs play pivotal roles in several biological processes.9 The physiological roles associated with
these enzymes are diverse, ranging from facilitators of plant immune defence mechanisms to
enzymatic breakdown of glucocerebroside and in treatment of Gaucher’s disease in humans.10, 11At
present, the tertiary structures of several BGLs have been determined. All of these BGLs share a
similar structural motif, and similar catalytic reaction mechanism.12, 13
Almond β-glucosidase activity has been well investigated. However studies investigating
correlation between its flexibility and activity is not as well understood as that between structure
and activity. Previous studies have investigated the dynamical nature of BGL upon substrate
binding, 14, 15 which provides little to no information regarding the essential motions responsible
for catalysis. Upon an inspection of molecular structure of the almond BGL four loop regions
(LRs) were found to be placed strategically over the catalytic barrel of the enzyme like a lid. We
conjecture that these LRs are involved in a gatekeeping mechanism to control enzymatic activity
at different temperatures. In the prsent study, essential structural dynamics of almond BGL was
correlated with its catalytic activity at different temperatures using steady state, circular dichroism
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(CD) and picosecond resolved fluorescence spectroscopy along with an all atom molecular
dynamics (MD) simulation, principal component analysis (PCA) and steered molecular dynamics
simulation (SMD).
Result and Discussion
Homology modelling
Almond BGL [GenBank accession number AFH35017] exhibited a low similarity with structures
within the PDB database based on the results of sequence analysis tools. The sequence search by
NCBI BLAST16 over structures within PDB revealed that BGL matches with cyanogenic β-
Glucosidase of Trifolium repens (1CBG) having 63% sequence identity. Accordingly, homology
modelling method was employed to construct a 3D-model for BGL. The amino acid sequence was
analysed using a set of known fold recognition online servers including PSI-BLAST,16 Phyre2,17
and HHpred.18 All servers commonly showed the cyanogenic β-Glucosidase from T.repens
(1CBG)19 with the highest identity and lowest E-value. The BGL structure was found to belong to
the same TIM barrel family of 1CBG: family 1 of glycosyl hydrolase. Consequently, 1CBG was
proven to be a suitable template structure for modelling the BGL structure. The 3D-model was
built for BGL sequence using the SWISS-MODEL server.20 The 1CBG protein structure was used
as a template.
The constructed 3D-model was submitted to different tools for quality evaluation. The backbone
conformation study by the PRO-CHECK web server based on the Psi/Phi Ramachandran plot
revealed that 90% of the residues were located in the most favourable region, 9.4% in the additional
allowed region, 0.4% in the generously allowed region and 0.2% residues in the disallowed regions
(Supplementary FigureS1). The evaluation by VERIFY-3D web server showed that 94.87% of the
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residues resides in a favourable environment having an average 3D-1D score of above 0.2.21
Moreover, the statistics of highly refined structures were compared using the ERRAT web tool to
study the overall quality of the model for non-bonded atomic interactions. The computed score by
ERRAT (94.51%) indicates that the model is acceptable in the normal range (above 50%). The
quality of the constructed model was confirmed based on the scores obtained from the above
evaluation tools. The summary of the model analysis are shown in Supplementary Table S1.
Figure 1(a) shows the homology model of almond BGL. The BGL model has similar
characteristics of other family members of 1 glycosyl hydrolases. The catalytic domain of the
enzyme (BGL) consists of alternating pattern of β-strands and α-helices that is a TIM barrel fold
[(α/β)8].22 The BGL structures mostly consist of a TIM barrel as a common folding motif.23
Evolutionarily, family 1 glycosyl hydrolases are dyad and Figure 1(b) shows the conserved
catalytic sites on the β-barrel (Glu424; Glu211) of BGL. The catalytic site on the β-barrel is
covered by four mostly unstructured regions guarding the entry gate of substrate as a lid. The
regions are marked as follows: LR1 (residue no: 77-100); LR2 (residue no: 227-242); LR3a
(residue no: 359-376); LR3b (residue no: 377-387); LR3c (residue no: 388-402) and LR4 (residue
no: 426-449) as shown in the figure 1(c) and (d).
Secondary structure analysis
Almond β-glucosidase is relatively more thermostable than other β-glucosidases of plant origin,24
including vanilla,25 grape,26 maize, 27 and tea leaves β-glucosidases.28 The thermal stability of BGL
was quantified by circular dichroism (CD) spectroscopy in the far UV region. The far UV CD
spectra of BGL were characterized by two minima at 208 nm and 220 nm (Supplementary
FigureS2).29 Upon deconvolution of the spectra insignificant changes in the secondary structure
was observed indicating to the absence of thermal denaturation within the temperature range of
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our study. Only 1.7% decrease in the α-helix, 5.5% increase in antiparallel β-sheet, increase of
0.3% parallel β-sheet, 0.6% β-turn and 0.4% increase in random coil was observed (Supplementary
Table S2a). The refolding of the BGL molecule was studied through gradual cooling of the enzyme
to 278K and subsequently, deconvolution of the CD spectra was found to manifest retention of the
enzyme native structure with 1.0% recovery of α-helix and 2.0% in antiparallel β-sheet
(Supplementary Table S2b). No significant changes in random coil, turns and parallel β-sheets
were observed (Supplementary Table S2b). Thus, from secondary structure analysis no appreciable
structural denaturation of the enzyme was noticed upon thermal treatments. Upon re-evaluation of
the CD spectra using BestSel CD deconvolution server similar trends in the results were obtained.
No significant change to the secondary structure was observed (Supplementary Table S2a & S2b),
indicating and reaffirming the thermal stability of the enzyme.
Enzymatic activity assay
Enzyme kinetics of BGL glucosidase activity were conducted in details at temperatures ranging
from 278K to 353K. The initial velocity of the glycoside hydrolysis by BGL was measured under
different rutin (substrate) concentrations (4–20 mM). Lineweaver-Burk plots were used to
determine the reaction kinetic parameters (Km, Vmax, kcat, kcat/Km). BGL belongs to the class of
glycohydrolases that catalyzes the hydrolysis of its natural substrate rutin into quercetin (Figure
2a and 2b). Figure 2c and 2d illustrate the effect of increasing temperature on the product formation
and catalytic efficiency. Thermal treatment was found to enhance the catalytic efficiency of the
enzyme (almond BGL) which becomes maximum at 333K, and further increase of temperature to
353K resulted in decreased catalytic efficiency relative to that at 333K. It is also noteworthy that
no discernible product formation was observed at temperature as low as 278K, while low product
formation and reduced catalytic efficiency was observed at a much higher temperature (353K).This
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is consistent with thermal stability of almond BGL over a wide range of temperature indicating to
formation of a stable enzyme-substrate complex even at a temperature as high as 353K. The
summary of the kinetics parameters are shown in Table 1.
Time-resolved Fluorescence Anisotropy
Molecular docking studies of ANS in presence of the enzyme (BGL) shows its’ molecular
interactions with the amino acid residues of BGL (Figure 3). The targeted docking found
interactions between the probe and the tryptophan residues (Trp396 and Trp475) at the catalytic
barrel of BGL, which was close to the active site. The dynamical flexibility of BGL was revealed
from time-resolved anisotropy decay (r(t)) of ANS30-34 in its complex with the enzyme at different
temperatures (Figure 4) over the range of 278K to 353K. A three exponential model was required
to obtain satisfactory fits to the polarization gated anisotropy decay, r(t). The fast rotational
correlation time (φ1) of ~100-400 ps corresponds to rapid local motion of the probe ANS bound to
Tryptophan (Trp-396 and Trp-475) residues, whereas the slower one (φ2) of 0.5-2.36 ns (Table 2)
is reflective of segmental motion of the neighbouring loop regions close to the active (catalytic)
site or the catalytic barrel. The much longer time constant (φ3 > 20 ns, Table 2) in the anisotropy
decay (Figure 4) corresponds to global tumbling motion of the overall probe-enzyme complex. A
monotonous decrease in both the correlation times (φ1 and φ2) was noted with rise in temperature.
In comparison to the lower temperature range (278K to 313K) significant decrease in φ1 and φ2
was noted at 333K or 353K due to increased mobility of the dye local environment that results
from significantly increased dynamical flexibility of the amino acid residues (Trp-396 and Trp-
475) and the loop regions close to the catalytic barrel. The significant decrease, especially in φ2, in
going from 313K to 333K indicates significantly increased dynamic flexibility of the loop regions
which likely facilitates substrate binding to the catalytic barrel resulting in enhanced catalytic
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efficiency at the higher temperature (333K). Upon further increase of temperature to 353K,
remarkable decrease in the rotational time constants was observed which is consistent with rapid
fluctuations of the amino acid residues and loop regions near the catalytic barrel which may
sometimes impede substrate entry into the catalytic barrel or lower the stability of the enzyme-
substrate complex.
Molecular dynamics (MD) simulation
The stability and mobility of the BGL structure was studied by subjecting the structure to 48 ns all
atom MD simulation at low (278K), moderate (313K), moderately high (333K) and high (353K)
temperatures. Supplementary Figure S3 shows the RMSD and Rg plot of BGL at different
temperatures. RMSD indicates the overall change in the geometry of the enzyme with respect to
its initial structure. From Figure S2 it becomes evident that after ~25 ns, the structure reaches an
equilibrium geometry. Further, there is a monotonous increase in the RMSD value with respect to
the temperature echoing the effect of temperature on flexibility of the enzyme. In order to have an
insight into the effect of temperature on the overall shape and compactness of the enzyme the
radius of gyration (Rg) was calculated. Rg is defined as the average distance of the collection of
atoms from their common centre of mass. Higher Rg values indicate looser packing of the protein
structure whereas lower values respond to a much compact structure of it. Figure S2 shows further
that there is no significant change in the overall shape and compactness of BGL over the course of
the simulation event corroborating our data from circular dichroism study. Furthermore, flexibility
in different parts of the enzyme (BGL) at different temperatures was assessed by calculating the
Root mean squared fluctuation (RMSF) which is a measure of the displacement of an amino acid
residue around its averaged position during a defined period of time and allows detection of the
regions of high flexibility in a protein. Figure 5 shows RMS fluctuations of BGL at different
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temperatures. It is observed from Figure 5a and 5b that the loop regions experience the most of the
residue fluctuations in BGL. Four loop regions in particular form the epicentre of residue
fluctuations: LR1, LR2, LR3, and LR4 (Figure 1) in the enzyme. These regions comprise of
residues 77-100 (LR1), 227-242 (LR2), 359-376 (LR3a), 377-387 (LR3b), 388-402 (LR3c), and
426-449 (LR4). All these regions experience fluctuations greater than 4Å whereas the regions
experiencing fluctuations lesser than 4Å were mostly found to be located in the binding site or the
catalytic barrel having conserved residues. Figure 5c shows fluctuations experienced by the LRs
at different temperatures. It is clearly evident that the LR1 undergoes monotonous increase in
fluctuations as does the LR2 with rise in temperature. However, the LR3 region shows a form of
synchronous fluctuations at its sub regions LR3a and LR3c while the LR3b remains rigid acting
like a pivot. The LR4 region however displays an unusual fluctuation. The LR4 region shows
increased fluctuations at 278K, the movement becomes fairly rigid at 313 and 333K, increasing
again at 353K. It is noteworthy that Figure 5c does not indicate the magnitude of fluctuations but
rather the effect of temperature change on the LRs with respect to control (non-LRs). The changing
pattern of fluctuations in the LRs indicate a possible pattern over the course of temperature range.
It should be noted that the LRs cover the catalytic barrel like a lid whose fluctuations likely
contribute to or control the catalytic activity of the enzyme.
Normal mode analysis
To obtain better insights and identify the overall pattern of motions of the entire protein Principal
component analysis (PCA) was used. The eigenvectors are calculated from the covariance matrix
of a simulation trajectory. The trajectories are filtered along each eigenvector to identify the
dominant motions during a simulation event. A large portion of the overall fluctuations of the
macromolecules observed from RMSF can often be accounted for by a few low-frequency
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eigenvectors with large eigenvalues. If the motions are similar, the eigenvectors and eigenvalues
coming from individual trajectories should be similar to each other. All atom Principal component
analysis was applied to study the major motions of BGL at different temperatures. Figure 6 and
Figure 7 display the porcupine plot of the first two eigenvectors and the mobility plots of BGL.
The mobility plot (Figure 7) clearly shows an initial increase in motion in the LR3 with a decrease
after 333K, which directly correlates with the catalytic activity of the enzyme. From the porcupine
plots (Figure 6) we can clearly visualize the major motions of the protein at different temperatures.
Figure 6 indicates that the LR region is the epicentre of major motions in the protein. At 278K the
LR4 region serves as the centre of major fluctuations while the other LR regions remain rigid. In
case of 313K major motions in the LR3 region are observed. The porcupine as well as the mobility
plots indicate the presence of three different motions at the LR3 region. The LR3A and LR3C
regions show a propeller motion; moving in clockwise direction, while the LR3B region remains
rigid acting like a pivot. The motion of the entire protein is clockwise in the first mode. In the
second mode, the motion of LR3 is not the primary motion and the protein as a whole moves in an
anti-clockwise direction relative to that of LR3. This suggests a possible change in the orientation
of the loop region covering the catalytic barrel making it more accessible to the incoming substrate.
The principal motions for BGL at 333K illustrates the clockwise propeller motion of the LR3
region, while the entire protein experiences a counter-clockwise motion. Both the modes in the
porcupine plot and the mobility plot indicate the presence of significant motions in the LR2 region.
The presence of new sites of prominent motion could aid in proper orientation of the catalytic site
for the approaching substrate, thereby, facilitating the enzymatic activity of BGL. The mobility
plot at 353K shows the presence of several new sites of motion (LR1 and LR2). From the porcupine
plots random and independent motions of different sites are visualized and the fluctuations of the
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LRs become highly inordinate and asynchronous which may affect the enzymatic activity at high
temperatures. [Visual information regarding the various modes and major motions are provided in
a Supplementary Video S1-4].
Steered molecular dynamics simulation
Study of the energy required to pull a substrate molecule (rutin) out of the binding site of BGL
(Figure 8a), gave further clarity to the notion of ease of substrate binding at higher temperature.
Following the methodology of steered molecular dynamics simulation (SMD) described in the
earlier work,35 it was observed (Figure 8b) that with increasing temperature less energy was
required to break the enzyme substrate interactions and pull the substrate out. 333K was found to
be the optimum temperature at which a meta-stable energy minimum was observed after a short
pull (Figure 8b). Molecular interaction studies at the minimum indicated compensating bond
formation and breakdown with the substrate (Figure 8c & 8d). No compensating interactions were
found for 353K, which resulted in the monotonous increase of the potential mean force (PMF)
(Figure 8b, 8e, 8f). Detailed analysis of the interacting residues found the presence of water-bridge
connecting Arg-325 to the hydroxylic group adjacent to the glycosidic bond at the meta-stable
state (Figure 8c & 8d). Our previous work had discussed the role of bulk and bound water
molecules in biological recognition and hydrolysis.36 The bound water molecule in the meta-stable
state may be involved in the catalytic activity of the enzyme. Table 3 lists the equilibrium distances
between the centre of mass of BGL and rutin. It was evident from the table that rutin (substrate)
can go further into the active site of BGL at 333K than any other temperature. Solvent exposure
of the substrate at different temperatures (Table 4) was also consistent with the finding.
Structure-Activity relationship
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Almond BGL causes the catalytic hydrolysis of the flavonoid glycoside Rutin to release Quercetin.
Temperature dependent enzymatic activity was monitored at temperature ranging 278K-353K. We
observed no product formation at temperatures as low as 278K. Increased product formation at
temperatures as high as 333K suggested the ease of the enzyme substrate complex formation. The
kinetics parameter were calculated from the Lineweaver-Burk plot. The catalytic efficiency
(kcat/Km) was a typical bell shaped curve, temperature optima at 333K, which was in agreement
with previous studies (Figure 2) 24. Loss of catalytic activity was observed at 353K, however the
thermal stability observed at this temperature pointed towards dynamical flexibility being the
possible architect of the structure-activity relationship.
The results of activity study point to the dynamical flexibility as the possible facilitator of
enzymatic activity at different temperatures. Studies by Banerjee and Pal, 37 Karplus et al 38 and
Min et al 39 found a direct correlation between picosecond to nanoscond timescale dynamics and
enzymatic activity. Picosecond resolved fluorescence anisotropy of the ANS-BGL complex
comprehensively shows a monotonous increase in dynamical flexibility of the protein with rise in
temperatures (Figure 4 & Table 2). The rise in temperature creates a more labile microenvironment
of the probe enhancing orientational relaxation of ANS. RMSD calculations further support the
activity-flexibility correlation being solely responsible in catalysis (Supplementary Figure S2).
While the RMSF studies identify four loop regions [LR1; LR2; LR3; LR4] as the possible regions
of high fluctuations (Figure 5). The loop regions (LRs) are placed strategically as a lid over the
catalytic barrel. Other regions of the enzyme experiences fluctuations of lesser magnitude than the
loop regions. Time-resolved anisotropy measurements and RMSF analysis as well as the SMD
simulations indicate to the possible gatekeeping function of the LRs where the change in
fluctuations of the LRs associated with their possible orientation and re-orientation at an optimized
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position dictates the entry of an incoming substrate at the active site of the catalytic barrel. Our
study also indicates that higher degree of fluctuations beyond the threshold level at 353K (Figure
4) likely cause the LRs to an unfavourable orientation for entry of the substrate at the active site,
leading to reduced catalytic efficiency (kcat/Km) (Figure 2).
Loop dynamics controls catalysis in almond β Glucosidase
The RMSF studies indicate towards four strategically placed LRs as the possible gatekeepers of
enzymatic activity at different temperatures. The overall fluctuations of the macromolecule
observed from RMSF cannot provide concrete information regarding major motions along the
trajectories of simulation. The trajectories need to be filtered along each eigenvector to identify
the dominant motions during a simulation event. Principal component analysis (PCA) of BGL at
different temperatures identified the LRs as the epicentre of dominant motions at different
temperatures. Our study also found that residues of the catalytic barrel experienced no major
fluctuations across all temperature ranges. Dynamical rigidity of the LRs at 278K barring LR4
likely results in a possible closed gate conformation checking the access of an incoming substrate
in the catalytic site and in consequence no enzymatic activity at the said temperature (Figure 6 &
7) [Supplementary Video S1] has been noted. The concerted motions of LR3 and rest of the
protein, along with the propeller motion of LR3 indicates to a change in orientation to an open gate
conformation at 313K (Figure 6 & 7) [Supplementary Video S2]. The open gate conformation
would allow access to the substrate resulting in enzymatic activity at 313K. The concerted motion
was maintained at 333K and new centres of prominent motions at LR1 and LR2 were observed
(Figure 6) [Supplementary Video S3]. These new sites of prominent motion could aid in the
optimum orientation of the lid LRs aiding in enhanced catalysis and turnover observed at 333K
(Table 1). Ease of substrate access at this temperature also reverberated in the least pulling force
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as observed in SMD simulations (Figure S4). At 353K there is a loss of concerted motions of the
LRs and fluctuations become independent of each other and random with several new sites of
major motions (Figure 6) [Supplementary Video S4]. This loss in the concerted motion of the LRs
and increased randomness may rationalize increased flexibility of the ANS-BGL complex
observed in polarization gated anisotropy measurements (Figure 4) and the fall in the catalytic
activity and turnover at 353K (Figure 2 & Table 1). The random fluctuations of the LRs may lead
to the inability or possibly dissociation of the substrate from the active site of the enzyme (BGL)
contributing to reduced catalytic efficiency.
Materials and Methods
Materials
Beta-Glucosidase (Prunus dulcis) (≥ 6U/mg), Rutin hydrate (≥94%), 8-Anilino-1-
napthelenesulfonic acid (ANS) (≥97%) were purchased from Sigma (Saint Louis, USA). Ethanol
(ACS) was purchased from Merck (Darmstadt, Germany). BGL and ANS solutions were prepared
in Phosphate buffer (10mM, pH 7.0) using water from Millipore system. Rutin solution was
prepared in ethanol.
Sample Preparation
The BGL-ANS complex for anisotropy analysis was prepared by mixing ANS (0.6µM) with BGL
(6µM) in phosphate buffer (pH 7.0) with continuous stirring for 5h at 4oC, which was filtered
extensively to remove the free probe from the solution 31.
Computational modelling of the enzyme
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The full length amino acid sequence of almond BGL was obtained from NCBI GenBank database
[Accession number: AFH35017.1]. Homology modelling was used to construct a 3D structure for
the protein using Trifolium repens cyanogenic beta glucosidase (1CBG) as template. SWISS-
MODEL server was used to build the 3D structure of the enzyme. Assessment of the constructed
3D model was done by the VERIFY-3D,40 PRO-CHECK,41, ERRAT,42, and ProSA-Web 43 web
servers.
Circular dichroism (CD) spectroscopy
Temperature dependent circular dichroism (CD) spectroscopy study was conducted with 1 μM
BGL solution using a quartz cuvette of 1 mm path length in a JASCO 815 spectrometer with a
temperature controller attachment (Peltier). The experimental temperature was gradually increased
from 278K to 353K and then decreased back to 278K. The deconvolution of the CD signals into
relevant secondary structure was done by CDNN software44 and were re-evaluated using BestSEl
CD deconvolution server. 45
Enzyme activity assay
Temperature-dependent (278K-353K) enzymatic activity studies were conducted with BGL
concentration 0.5 μM and the concentrations of the substrate Rutin (λmax ~ 358 nm) was varied in
the range 4-20 mM. The substrate was cleaved to produce Quercetin (λmax ~ 374 nm), the
absorbance of which was monitored in Shimadzu Model UV-2600 spectrophotometer with an
attachment for temperature dependent studies (TCC 240 A). The Michaelis-Menten kinetics
parameters were determined from the Lineweaver-Burk plot by a nonlinear least square fitting
software. 46
Molecular docking
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Molecular docking studies were performed to identify the binding site of ANS and rutin in BGL.
The ligand (ANS & rutin) structures were obtained from the PubChem database. Targeted docking
was performed at the active site using AutoDock Vina.47 The molecular interactions were viewed
using Schrodinger Maestro 2018-1 (Academic release).
Polarization-gated Anisotropy Measurements
Time resolved fluorescence transients were measured in a LifeSpec-ps picosecond diode laser-
pumped fluorescence spectrophotometer from Edinburgh Instruments (Livingston, U.K).
Picosecond excitation pulses from the picoquant diode laser was used at 375 nm for excitation of
ANS with an instrument response function (IRF) of 60 ps with an attachment for temperature
dependent studies [temperature range: 278K-353K]. A microchannel-plate-photomultiplier tube
(MCP-PMT; Hammamatsu Photonics, Kyoto, Japan) was used to detect the photoluminescence
from the sample after dispersion through a monochromator. Our time-resolved instrument provide
a time resolution of 20 ps which is one third of the instrument response (IRF) for detection of time
constants after the de-convolution of the IRF. Time-resolved anisotropy, r(t), was determined from
the following equation
IVV(t) ― G.IVH(t)
r(t) =
IVV(t) + 2.G.IVH(t)
Where IVV(t) and IVH(t) are parallel and perpendicular polarized fluorescence decays of the dye,
respectively, recorded using vertically polarized excitation light. G is an instrument and
wavelength dependent correction factor to compensate for the polarization bias of the detection
system and its magnitude was obtained by a long tail matching technique.48, 49 The anisotropy
3
∑
decays were fitted using a multi-exponential decay model
where 푟
푟(푡) = 푟0 푖 = 1훼푖exp( ― t/휃푖)
0
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is initial anisotropy, θ is are the rotational correlation times of the fluorescence probe (ANS)
hinting to dynamical flexibility of the probe environment in its complex with protein (BGL) 50.
Classical molecular dynamics (MD) simulation
The temperature dependent dynamical behaviour of BGL was further studied by molecular
dynamic (MD) simulation. The constructed BGL structure was subjected to energy minimization
by Schrodinger Maestro 2018-1(Academic release). Simulations were done at temperatures 278K,
313K, 333K and 353K. Protein molecule was placed in the centre of a periodic boundary box with
a minimum distance of 10 Å from each side. Then, the protein was solvated with pre-optimized
simple point-charged (SPC) water molecules. Additionally, the system was charge neutralized by
adding necessary counter ions (seven Na+) A five step relaxation protocol was used to equilibrate
the entire system 51. For the final runs, normal pressure and a defined temperature were used and
no particles were restrained. The ensemble type was NPT with a constant number of particles,
normal pressure (1 bar) and a defined temperature. Nosé-Hoover thermostat and Martyna-Tobias-
Klein barostat maintained the temperature and pressure of the system, respectively. MD was run
in OPLS 2005 force field 52 with a short range Coulombic interaction cut-off of 9 Å. Long range
coulombic interactions were handled by smooth particle mesh Ewald method with a Ewald
tolerance of 10-9. The simulations were run for 48 ns at the said temperatures. Overall structural
changes in the protein with time was computed in terms of root mean square deviation (RMSD).
Radius of gyration (Rg) and residue-wise fluctuations (RMSF) were also computed from the
simulation trajectory.
Normal mode analysis
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Reduction of dimensionality of the MD simulation data helps in identifying the configurational
space in which anharmonic motion takes place. Principal components analysis (PCA) takes the
trajectory of a molecular dynamics simulation and extracts the dominant modes in the motion of
the molecule.53-55 These dominant motions correspond to correlated vibrational modes or
collective motions of groups of atoms in the normal modes analysis. The direction and amplitude
of the dominant motions along a simulation trajectory by Principal component analysis (PCA) was
computed by Normal Mode Wizard in VMD 1.9.3 program 56 using ProDy57. Porcupine plots and
mobility plots for the respective temperature was generated by the program showing a graphical
summary of the motions along the trajectory. The Supplementary Video of the principal
component analysis were also prepared in VMD. 56
Steered molecular dynamics
Steered molecular dynamics35 was performed in Schrodinger Maestro 2018-1 (Academic release)
using the metadynamics module. Rutin bound BGL as obtained by molecular docking, was
subjected to MD simulation following the previously mentioned protocol at four different
temperatures. Then, starting with the temperature equilibrated substrate (rutin) bound BGL
conformations, steered dynamics was performed to probe the energy required to pull the substrate
out of the active site. Therefore, the distance from the centre of mass of BGL and the centre of
mass of rutin was gradually increased. A cut-off value for the subsequent increase in the energy
was set to be 20 kcal/mol. All the other parameters were same as the classical MD simulation.
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Conclusion
The 3D-model and secondary structure analysis of an enzyme almond BGL illustrated various
remarkable characteristics of this glycosyl hydrolase. The structure of this thermostable enzyme
consists of four loop regions (LRs) placed strategically over the catalytic barrel as a lid. The
concerted motion of these LRs likely acts as a gatekeeper of catalytic activity of the enzyme at
different temperatures, and consequently, its ability to be active at moderately high temperatures.
The findings of this study can provide a background for future efforts to enhance enzymatic
capabilities through rational design of recombinant enzymes.
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CONFLICT OF INTEREST
The authors declare there is no conflict of interest in this work.
ACKNOWLEDGMENT:
The authors thank DST-SERB, India, for financial grants, EMR/2016/004698. The authors
gratefully acknowledge the financial assistance provided by UGC (F.PSW-137/15-16 ERO). The
authors also thank DBT (WB)-BOOST scheme for financial grant, 339/WBBDC/1P-2/2013.
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Tables
Table 1. Temperature-Dependent Kinetics of BGL Catalyzed Hydrolysis of Rutin
Km (M)
Vmax (μM/s)
0.15±0.007
0.4±0.020
Kcat (s-1) kcat/Km (M-1s-1)
Temperature (K)
293K
(4.7±0.23)×10-5
(5.1±0.25)×10-4
(7.9±0.39)×10-4
(1.27±0.06)×10-3
(1.04±0.05)× 10-3
0.3±0.015
0.8±0.040
638±31.90
1311±65.55
1367±68.35
2362±118.10
1423±71.15
303K
313K
333K
353K
0.54±0.027 1.08±0.054
1.5±0.075
3.0±0.150
1.4±0.070
0.74±0.037
Table 2. Time-resolved Anisotropy of ANS in BGL at Different Temperatures
Temperature(K)
r0
φ1/ns
φ2/ns
φ3
278K
293K
303K
313K
333K
353K
0.37
0.38
0.38
0.38
0.400 ± 0.040 (21%)
0.330 ± 0.030 (22%)
0.206 ± 0.020 (29%)
0.185 ± 0.015 (34%)
2.36 ± 0.20 (25%) > 20 ns
2.14 ± 0.20 (23%) > 20 ns
2.03 ± 0.15 (26%) > 20 ns
1.61 ± 0.15 (27%) > 20 ns
0.81 ± 0.08 (26%) > 20 ns
0.54 ± 0.05 (33%) > 20 ns
0.387 0.140 ± 0.010 (28%)
0.39
0.110 ± 0.010 (27%)
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Table 3. Distance of Center of Mass of the Protein (BGL) to the Center of Mass of the
Ligand (Rutin)
Temperature (K)
Distance (Å)
12.02
278
313
333
353
11.05
9.78
10.46
Table 4. Solvent Exposure of the Ligand (Rutin) in Terms of Interaction Energies with
Water
Temperature (K)
Energy (kcal/mol)
-35.47
278
313
333
353
-31.70
-31.20
-53.35
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FIGURES
Figure 1 Schematic showing the build structure of almond Beta-Glucosidase (BGL). (a) The
modelled 3D structure. (b) The stick representation showing the conserved active site Glutamate
residues. (c) Showing the four loop region (LRs). The coloured regions represents each of the LRs,
LR1 [Blue]; LR2 [Green]; LR3 [Yellow]; LR4 [Orange]. (d) Space filling model showing the
strategic positioning of the LRs along the active site [AS], LRs represented by the same colour
scheme.
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Figure 2 Schematic representation of temperature dependent enzyme catalysis. (a) Simple model
outline of the reaction catalysed by BGL, the flavonoid glycoside Rutin hydrolysed to produce
Quercetin. (b) Absorbance spectra of the substrate Rutin and product Quercetin during catalysis.
The inset representing normalised absorption spectra of the same. (c) Product formation at
different representative temperatures showing enhanced product formation at 333K. (d) Showing
the effect of temperature on the catalytic efficiency (kcat/Km) of BGL. The enzyme showed
enhancement of catalytic efficiency with increased temperature generating a bell shaped curve.
Temperature optima was observed at 333K. Fall in efficiency was observed beyond 333K.
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Figure 3 Ribbon representation of docked conformation of ANS-BGL complex. (a) Molecular
docking representation of ANS-BGL complex, ANS [shown in pink] was found to be bound at the
catalytic barrel of BGL. (b) ANS docked at the catalytic barrel. The fluorescence probe was found
to interact with two Tryptophan residues (Trp396; Trp475). (c) Molecular interaction diagram
between ANS and Tryptophan residues via π-stacking [shown in green lines] at the catalytic barrel.
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Figure 4. Temperature dependent anisotropy decay
29