Organic &

Biomolecular Chemistry
View Article Online
PAPER View Journal | View Issue

The role of conserved arginine in the GH70 family:

a computational study of the structural features
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.

Cite this: Org. Biomol. Chem., 2019,

17, 6269 and their implications on the catalytic mechanism
of GTF-SI from Streptoccocus mutans†
Leslie Sánchez,a Fernanda Mendoza, b Joel B. Alderete, c

Verónica A. Jiménez b and Gonzalo A. Jaña *b

Received 7th May 2019,
Accepted 5th June 2019
DOI: 10.1039/c9ob01055f
rsc.li/obc
In this work, molecular dynamics and QM/MM calculations were employed to examine the structural
and catalytic features of the retaining glucosyltransferase GTF-SI from the GH70 family, which partici-
pates in the process of caries formation. Our goal was to obtain a deeper understanding of the role of
R475 in the mechanism of sucrose breakage. This residue is highly conserved in the GH70 family and so
far there has been no evidence that shows what could be the role of this residue in the catalysis per-
formed by GTF-SI. In order to understand the structural role of R475 in the native enzyme, we built full
enzyme models of the wild type and the mutants R475A and R475Q. These models were addressed by
means of molecular dynamics simulations, which allowed the assessment of the dynamical effect of the
R475 mutation on the active site. Then, representative structures were chosen for each one of the
mutant models and QM/MM calculations were carried out to unravel the catalytic role of R475. Our
results show that the R475 mutation increases the flexibility of the enzyme, which triggers the entrance
of water molecules in the active site. In addition, QM/MM calculations indicate that R475 is able to
provide a great stabilization to the carboxylate moiety of the acid/base E515, which is an essential
characteristic favoring the proton transfer process that promotes the glycosidic bond breakage of
sucrose.

1. Introduction Glucansucrases are the members of the Glycoside Hydrolase

Glucansucrases (GSs) or glucosyltransferases (GTFs) are extra-
cellular enzymes produced by lactic acid bacteria of the genus
Leuconostoc, Streptococcus or Lactobacillus. From sucrose,
they catalyse the synthesis of high molecular weight α-glucans
through the formation of glycosidic bonds between glucose
units. So, depending on the enzyme specificity, a wide range of
α-glucans can be produced, varying in terms of size, glycosidic
linkage, composition and degree of branches.1–3 This struc-
tural variability results in a wide range of physicochemical pro-
perties, which may be useful for different applications.4,5
a
Doctorado en Fisicoquímica Molecular, Universidad Andres Bello, Republica 275,
Santiago, Chile
b
Departamento de Ciencias Químicas, Facultad de Ciencias Exactas,
Universidad Andres Bello, Sede Concepción, Autopista Concepción-Talcahuano 7100,
Talcahuano, Chile. E-mail: gonzalo.jana@unab.cl
c
Instituto de Química de Recursos Naturales, Universidad de Talca,
Avenida Lircay s/n, Casilla 747, Talca, Chile
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9ob01055f
70 (GH70) family and together with the enzymes from GH13
and GH77 families, they form the clan GH-H, sharing similar
mechanisms and structural and evolutionary characteristics.
GSs catalyse the glucose transfer reaction via an α-retaining
double displacement mechanism, which requires three amino
acid residues (catalytic triad) composed of two aspartic acids
and one glutamic acid, similar to that of amylosucrases.6
The proposed catalytic mechanism starts with the binding
of the sucrose substrate and the cleavage of the α(1 → 2) gly-
cosidic linkage between the glucosyl and fructosyl moieties
yielding the Covalent β-Glucosyl Enzyme (CGE) intermedi-
ate. This occurs via an oxocarbenium ion-like transition
state, this step being the rate-limiting step. As shown in
Fig. 1, the first stage (glycosidic breakage) occurs through
the concerted attack of a nucleophilic residue (Asp) on the
anomeric carbon atom of sucrose and the simultaneous
protonation of the glycosidic oxygen by a glutamic acid. In
the second step (transglucosylation), the leaving group
(fructose) is exchanged by the new acceptor sucrose (R-OH),
which gets deprotonated by a glutamate residue acting
as a base. This activated acceptor attacks the covalent

This journal is © The Royal Society of Chemistry 2019 Org. Biomol. Chem., 2019, 17, 6269–6276 | 6269

Paper
Fig. 1 General double displacement mechanism proposed for the synthesis of α-glucans by GSs.
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.
intermediate in a concerted step and forms the final
α-glucosylated product via another oxocarbenium ion-like
transition state.
The catalytic triad is strictly conserved for all the members
of the GH70 family with known primary structures to date, and
their mutation leads to inactive enzymes.5,7,8 Actually, our
group, considering a full enzyme model of GTF-SI from
Streptococcus mutans, has recently published a paper which
describes in detail how this enzyme catalyses the first stage of
the mechanism of glucan formation through QM/MM calcu-
lations.9 Our results suggest a concerted asynchronous mecha-
nism for sucrose hydrolysis in which the first event corres-
ponds to the glycosidic bond breakage assisted by Glu515.
Then, the nucleophilic residue Asp477 attacks the glucosyl
moiety of sucrose leading to the formation of the CGE inter-
mediate. The TS was found to be strongly stabilized by the
hydrogen bond (OG⋯HE2⋯OE2) formed between the partially
transferred proton to glycosidic oxygen (OG) of the fructosyl
moiety from the Glu515 residue, which according to the dis-
tance OG⋯OE2, charges and 2nd-order perturbation analysis
can be considered as a short-strong hydrogen bond. This
hydrogen bond was not observed in a reduced model of the
active site that considered the catalytic triad, because the
proton is not transferred from Glu515 to glycosidic oxygen of
the sucrose. Therefore, we postulated that the positive charge
of one conserved arginine in the active site of the GH70 family
(R475 in GTF-SI), localized between D477 and E515, could
assist the proton transfer from E515 to the fructosyl leaving
group by stabilizing the growing negative charge on the gluta-
mate moiety by means of long-range electrostatic interactions.
However, this important role attributed to R475 has not been
validated until now, since there are no theoretical or experi-
mental studies on the GTF-SI from Streptococcus mutans which
account for the role of the conserved arginine in the enzymatic
catalysis.
Therefore, in order to obtain deeper insight into the role of
the conserved arginine, molecular dynamics (MD) simulations
were carried out for wild type and mutated GTF-SI enzyme
models. Moreover, for all mutated systems, QM/MM calcu-
lations were carried out to obtain the energy profiles, which
give us an account of the effect of single mutation on the
energy barriers.
6270 | Org. Biomol. Chem., 2019, 17, 6269–6276
View Article Online
Organic & Biomolecular Chemistry
2. Methods
2.1 Model construction
In this study, a GTF-SI·sucrose complex optimized at a QM/
MM level was used as the starting point to build the models.
This complex was obtained from a previous study in which the
crystal structure of GTF-SI (PDB code 3AIE) was aligned with
the GTF180-ΔN (PDB code 3HZ3) crystal structure, in accord-
ance with the methods described earlier.14 Herein, this model
will be named a wild type or native system. From the wild type
model, two new systems were built: R475A and R475Q. The
mutations were made with the mutator plugin of VMD soft-
ware10 and the PROPKA method was applied to determine the
protonation states of all ionizable residues at pH = 6.0, as was
described previously.9
2.2 Molecular dynamics simulations
All MD simulations were performed using the NAMD
program,11 with the CHARMM36 all-atom force field12–14 for
protein atoms, the TIP3P model for water molecules, and the
CHARMM36 all-atom carbohydrate force field for the disac-
charide and its glycosidic linkage.15 Periodic boundary con-
ditions were imposed and electrostatic interactions were
directly calculated with a cutoff of 12 Å, whereas long-range
electrostatic effects were taken into account by the Particle
Mesh Ewald (PME) summation method. van der Waals inter-
actions were treated with the use of a switch function starting
at 10 Å and turning off at 12 Å. MD simulations were carried
out with a standard protocol for each system. The three
models were solvated with a water box (85 × 114 × 87 Å3) cen-
tered at the geometrical center of the protein–substrate
complex. These dimensions of the box ensure that all protein
atoms are at least 10 Å away from the edges of the box. The
resulting systems have a negative net charge that was neutral-
ized by sodium cations initially positioned at least 5 Å away
from the protein surface. The final models contain 78837,
78821 and 78828 atoms in the wild type, R475A and R475Q,
respectively. Then, to remove close contacts and the highly
repulsive orientation of the initial protein–solvent system, we
first performed 1000 steps of energy minimization using the
conjugate gradient algorithm. From the resulting configur-
ation, MD simulations of 6 ns with a 1 fs time step were
This journal is © The Royal Society of Chemistry 2019
 View Article Online

Organic & Biomolecular Chemistry Paper

carried out to equilibrate the system in a NVT ensemble.

Equilibration was performed using positional restraints on the
protein and substrate starting with a force constant of 30
kcal mol−1 Å−2 that gradually decreased by 5 kcal mol−1 Å−2
every 500 ps until all positional restraints were removed. After
equilibration at 300 K, a 100 ns production period of MD simu-
lations was run using an integration time step of 2 fs in combi-
nation with the SHAKE algorithm for the O–H bonds of water
molecules. These simulations were run using the isothermal–
isobaric ensemble (NPT) at constant temperature and pressure
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.

using the NAMD Nosé–Hoover implementations.16–18

2.3 Quantum mechanics/molecular mechanics (QM/MM)

calculations
After the MD production simulations, representative structures
of R475A and R475Q models were selected to continue with
the QM/MM calculations. The water box was reduced to a
water sphere in a ratio of 25 Å around the glycosidic oxygen Fig. 2 The Reaction Coordinate (RC) has been defined as RC = d (C1 − OG)
(OG). The two models (R475A and R475Q) were partitioned − d (C1 − OD1) − d (HE2-OG). The labels of several atoms are shown and
into a Quantum Mechanics (QM) part defined by 67 atoms referenced throughout the Results and discussion section.

(link atoms are not included): 45 atoms of sucrose, the side

chain of the nucleophile D477 (6 atoms), the side chain of the
nucleophile D588 (6 atoms), and the side chain of the acid
E515 (10 atoms). Three link atoms were used, each one of
them located along the Cα-QM of the D477, D588 and the
D515, and the total charge of the QM subsystem was −2. The
geometry of the QM subsystem was described by the BP86/
6-31G(d) method, while the Molecular Mechanics (MM) part
was described using the CHARMM36 all-atom force field12–14,
and an electronic embedding scheme was used for the
QM/MM treatment. CHARMM19,20 and Q-Chem program
packages21–23 were employed to carry out the calculations.
It is worth mentioning that the BP86 functional has been
successfully tested for carbohydrate-related enzymes showing a
good compromise between computational efficiency and
accuracy.9,24,25 Thus, initially the QM part was frozen and the
rest of the system was optimized using a gradient criterion of
0.005 kcal mol−1 Å−1. Then, we defined an active region of
25 Å radius centered at the glycosidic oxygen. Every residue
within the active region was set free while the remaining resi-
dues were frozen during the optimizations. It was found that
this partial freezing method does not affect the energy bar-
riers.26 The procedure afforded an optimized reactant structure
that was used as the starting point for the initial reaction path
calculation using an asymmetric Reaction Coordinate (RC)
that allowed the conversion of the reactant to the CGE inter-
mediate (Fig. 2). Transition states (TS) were searched for the
reaction path obtained using the Conjugate Peak Refinement
(CPR) algorithm27 as implemented in the TREK module of the
CHARMM program, and the TS obtained from CPR were
characterized through a frequency calculation using QCHEM.
Finally, in order to correct the calculated energies, single
point energy calculations were carried out for every reaction
path using the Hybrid-Generalized Gradient Approximation
functional with the nonlocal correlation (H-GGA) ωB97X-V28
functional together with the 6-311++G(d,p) basis set. This
functional has proved to describe very well different molecular
properties.29
3. Results and discussion
Fully atomistic MD simulations and QM/MM calculation were
employed to investigate the role of the conserved arginine
residue R475 in the GH70 family, for which there is no infor-
mation about its structural or catalytic role. To this purpose,
the R475 residue of GTF-SI from Streptococcus mutans was
mutated for neutral residues, alanine and glutamine, provid-
ing three systems: wild type, R475A and R475Q. These compu-
tational experiments were aimed at providing structural and
electronic information about the role of R475 in GTF-SI.
3.1 Molecular dynamics simulations
As mentioned earlier, in GTF-SI from the GH70 family, the
sucrose interacts through hydrogen bonds with three catalytic
residues, which are essential for the catalysis: the nucleophile
(D477), acid/base catalyst (E515) and transition state stabilizer
(D588).9 Therefore, in order to enhance the sampling of cataly-
tically competent conformations, these interactions should be
maintained throughout the MD simulation.30 Thus, the dis-
tance between the glycosidic oxygen (OG) from sucrose and the
hydroxyl group of E515 was restrained to 2.0 Å using a harmo-
nic potential with a force constant of 50 kcal mol−1 Å−2, in a
similar way to what has been done in other studies.6
RMSD analysis of the three simulated systems (wild type,
R475A, and R475Q) indicates that system equilibrations were
achieved during the first 10 ns of simulation (data provided in
the ESI†). Consequently, trajectory analysis was carried out
during the last 90 ns of simulation.

This journal is © The Royal Society of Chemistry 2019 Org. Biomol. Chem., 2019, 17, 6269–6276 | 6271
 View Article Online

Paper Organic & Biomolecular Chemistry

Our results show that the mutation of R475 leads to the

entry of water molecules into the active site cavity of the
mutants R475A and R475Q. Actually, the average number of
water molecules within the active site of the wild type, R475A
and R475Q is 3, 21 and 13, respectively (data provided in the
ESI†). Solvent accessible surface area (SASA) calculations allow
us to verify that the single mutation of R475 helps to generate
a wider accessible surface near the catalytic site (Fig. 3A),
which explains the entrance of a larger number of water mole-
cules in the mutated systems. These results can be rationalized
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.

considering the alanine size, which is considerably smaller

than that of arginine or glutamine and therefore allows the
entrance of more water molecules than that observed for the
WT or the R475Q models. The flexibility introduced by this
single mutation is revealed by RMSF calculations (Fig. 3B),
which show that the sequence residues 530–540 and 835–846
from catalytic domain A present a higher mobility when com-
paring the mutants R475A and R475Q with the WT. In this
regard, the analyses of the R475A mutant enabled us to
observe an increase of ∼20 water molecules in the active site,
in contrast to that observed in the wild type, thus suggesting
an important structural effect of R475, as shown in Fig. 4.

Fig. 4 Representative snapshots of GTF-SI models from MD simulations

are depicted for A: R475A mutant and B: wild type enzyme. Water mole-
cules are represented as balls and sticks.

Moreover, it is interesting to mention that the mutant

models show that at least two water molecules are mimicking
the interactions set up by R475 in the WT, as shown in Fig. 5.
For the R475A model, one water molecule interacts with both
2OH and D477, while the second water molecule interacts with
E515. In a similar way, the R475Q model also exhibits a water
molecule simulating the interaction between R475 and D477.
These findings confirm the important electrostatic role of the
R475 residue in the active site, because when this residue is
mutated water molecules immediately mimic their interactions
with the catalytic residues and sucrose. Thus, these new find-
ings suggest that the mutation of R475 provokes relevant struc-
tural changes in the enzyme, such as an increase of water
molecules in the active site. Therefore, the larger number of
water molecules at the active site observed in the mutants indi-
cates that the R475 mutation could trigger the hydrolysis of
sucrose.

3.2 QM/MM calculations

MD simulations by themselves do not allow understanding
Fig. 3 A: Solvent accessible surface area (SASA) and B: RMSF what would be the catalytic implications of mutating the R475
fluctuation. residue by alanine or glutamine. Consequently, to address this

6272 | Org. Biomol. Chem., 2019, 17, 6269–6276 This journal is © The Royal Society of Chemistry 2019
 View Article Online

Organic & Biomolecular Chemistry Paper

Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.

Fig. 5 Representative snapshots obtained from MD simulations for the three models: wild type, R475A and R475Q.

issue, reaction paths were explored for R475A and R475Q by

means of QM/MM calculations. It is worth noting that the wild
type enzyme was not addressed in this work, because it was
already studied in a previous study, where an energy barrier
of 16.4 kcal mol−1 was obtained, in agreement with the
experimental results. Furthermore it was hypothesized that
R475 might assist in the proton transfer from E515 to the fruc-
tosyl (leaving) group by stabilizing the growing negative charge
on the glutamate moiety.9
Initial reactant structures used for the reaction path
exploration were selected from the MD simulations carried out
for each model (R475A and R475Q) according to the geometri-
cal parameters that are consistent with suitable catalytic con-
formations: (1) the distance of the nucleophilic aspartate to
the anomeric carbon should be close to 3.0 Å and (2) the dis-
tance between the acid/base residue E515 and the OG should
Fig. 6 Potential energy profiles (kcal mol−1) that schematically describe
not be longer than 2.0 Å. the CGE formation.
The initial guess reaction paths (data provided in the ESI†)
for the mutants were obtained through an asymmetric reaction
coordinate, obtaining energy paths consistent with only one
reaction event to describe the formation of the CGE intermedi- gered the entrance of water molecules to the active site. Two of
ate, in a similar way to what was found in the wild type.9 This these water molecules keep interacting with the catalytic resi-
reaction event is associated with a concerted mechanism, but dues D477 and E515 during the whole reaction path that leads
asynchronous, in which the glycosidic bond is being simul- to the CGE (Fig. 7A), which resembles the interactions that
taneously broken as the proton transfer occurs, followed by the R475 established in the WT enzyme with these catalytic resi-
nucleophilic attack. Thus, based on this guess reaction path, dues. In fact, the distance between the water molecule that
the Minimum Energy Path (MEP) was built using the CPR interacts with E515 decreases from 1.88 to 1.83 Å as the reac-
algorithm and the energy of the stationary points was cor- tant evolves to the TS structure, in a similar way to R475
rected at the ωB97X-V/6-311++G (d,p) level. The results show approaching E515 when transferred from the reactant to TS in
(Fig. 6) that when R475 is mutated, the calculated energy bar- the native enzyme. An analogous interaction with the one
riers for the mutant models are 25.7 kcal mol−1 (R475A) and described between the water and the acid E515 has been
31.5 kcal mol−1 (R475Q), which reveal an important stabilizing reported in another work30 as a very important one, since this
effect for this residue. Taking into account the energy barrier hydrogen bond lowers activation barriers by stabilizing the
reported for the WT enzyme9 (16.4 kcal mol−1), the mutation proton transfer involved in the HPA glycosidase mechanism
of R475 triggers an increase of the energy barriers by 9.3 and from GH13. So far, it can be concluded that to favor the proton
15.1 kcal mol−1 for R475A and R475Q, respectively. transfer from the acid E515 to the fructosyl group an inter-
Although the energy barrier rises in both mutant models, action appears to be necessary that can stabilize the growing
unraveling the difference in the energy barriers obtained for negative charge on the glutamate moiety, which is exerted by a
R475A and R475Q could help to gain a deeper insight into water molecule in this model. In addition, when the charge of
how R475 plays the proposed catalytic role. As was mentioned this water molecule is switched off the energy barrier increases
in the previous section, the mutation of R475 by alanine trig- by ∼3 kcal mol−1, indicating the relevance of stabilizing the

This journal is © The Royal Society of Chemistry 2019 Org. Biomol. Chem., 2019, 17, 6269–6276 | 6273

Paper
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.
Fig. 7 Molecular representations of the stationary points along the reaction path for the full enzyme models. The interactions established by the
water molecules and the catalytic residues are depicted by dashed lines in: (A) R475A and (B) R475Q.
glutamate moiety during the proton transfer. Actually, the
R475Q model comes to give additional support to this hypoth-
esis since, as can be seen in Fig. 7B, there is no water molecule
interacting with E515 along the reaction path, which entails a
significantly larger energy barrier for this model with regard to
the R475A mutant and native enzyme.
Furthermore, on the basis of geometrical parameters the
effect of the water molecule on the proton transfer is also
revealed when comparing the TS structure in R475A and
R475Q (Fig. 7). In the former case, it can be observed that the
proton transfer process has been completed and the glutamate
base is 1.48 Å away from the transferred proton. In the case of
R475Q, the proton is not fully transferred because it is located
at 1.36 and 1.13 Å from the glutamate base and the OG,
respectively.
On the other hand, the reaction energies associated with
the CGE formation are 9.7 and 23.8 kcal mol−1 in the R475A
and R475Q models, respectively, which suggest that the
mutation of R475 entails the destabilization of the CGE inter-
mediate. This effect is more pronounced in R475Q, which is
likely because there are no stabilizing interactions of E515, in
contrast to that observed in R475A (Fig. 7).
4. Conclusion
In this work, we combined MD and QM/MM calculations to
unravel the role of the conserved residue arginine located at
the active site of the members of the GH70 family. All results
6274 | Org. Biomol. Chem., 2019, 17, 6269–6276
View Article Online
Organic & Biomolecular Chemistry
together suggest that R475 not only has a key stabilizing effect
on the acid/base E515 during the proton transfer process, but
also plays a structural role in maintaining the active site fea-
tures suitable for the catalysis.
The MD simulations carried out for the mutant models
allowed the exploration of the structural effect of mutating
R475. The R475A model shows how the introduction of a sig-
nificantly smaller residue such as alanine increases the flexi-
bility of the residue sequence 530–540 and 835–846 from
domain A, which triggers the entrance of water molecules in
the active site. In the case of mutating R475 by glutamine,
which is a bigger residue than alanine, both the flexibility and
the water channel are observed but for a shorter period of
time.
Moreover, QM/MM calculations have shown the relevance
of the stabilization that provides R475 on the carboxylate
moiety of the acid/base E515, which is an essential character-
istic favoring the proton transfer process that promotes the gly-
cosidic bond breakage. Thus, we propose that R475 is essential
for both the structural stability of the active site and the cataly-
sis, providing a suitable environment for the chemical process.
Finally, although the role of R475 has not been evaluated
experimentally, our findings suggest that the mutation of con-
served arginine could trigger an entrance of water molecules
into the active site, implying that GTF-SI could lose its tranglu-
cosidase activity favoring the hydrolase activity. Therefore, we
hope that this new knowledge can help the experimentalists of
this field to further research into the structural and catalytic
roles of this conserved residue of the GH70 family.
This journal is © The Royal Society of Chemistry 2019

Organic & Biomolecular Chemistry
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors acknowledge the financial support from Grant
FONDECYT No. 1150704.
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.
References
1 H. Leemhuis, T. Pijning, J. M. Dobruchowska,
B. W. Dijkstra and L. Dijkhuizen, Biocatal. Biotransform.,
2012, 30, 366–376.
2 H. Leemhuis, T. Pijning, J. M. Dobruchowska, S. S. van
Leeuwen, S. Kralj, B. W. Dijkstra and L. Dijkhuizen,
J. Biotechnol., 2013, 163, 250–272.
3 M. I. Osorio, M. A. Zuniga, F. Mendoza, G. A. Jana and
V. A. Jimenez, Proteins: Struct., Funct., Bioinf., 2019, 87, 74–80.
4 S. Puanglek, S. Kimura and T. Iwata, Carbohydr. Polym.,
2017, 169, 245–254.
5 J. Gangoiti, T. Pijning and L. Dijkhuizen, Biotechnol. Adv.,
2018, 36, 196–207.
6 G. P. Pinto, N. F. Bras, M. A. S. Perez, P. A. Fernandes,
N. Russo, M. J. Ramos and M. Toscano, J. Chem. Theory
Comput., 2015, 11, 2508–2516.
7 X. F. Meng, J. Gangoiti, Y. X. Bai, T. Pijning, S. S. Van
Leeuwen and L. Dijkhuizen, Cell. Mol. Life Sci., 2016, 73,
2681–2706.
8 X. F. Meng, J. Gangoiti, N. de Kok, S. S. van Leeuwen,
T. Pijning and L. Dijkhuizen, Food Chem., 2018, 253, 236–
246.
9 G. A. Jana, F. Mendoza, M. I. Osorio, J. B. Alderete,
P. A. Fernandes, M. J. Ramos and V. A. Jimenez, Org.
Biomol. Chem., 2018, 16, 2438–2447.
10 W. Humphrey, A. Dalke and K. Schulten, J. Mol. Graphics
Modell., 1996, 14, 33–38.
11 J. C. Phillips, R. Braun, W. Wang, J. Gumbart,
E. Tajkhorshid, E. Villa, C. Chipot, R. D. Skeel, L. Kale and
K. Schulten, J. Comput. Chem., 2005, 26, 1781–1802.
12 A. D. MacKerell, D. Bashford, M. Bellott, R. L. Dunbrack,
J. D. Evanseck, M. J. Field, S. Fischer, J. Gao, H. Guo, S. Ha,
D. Joseph-McCarthy, L. Kuchnir, K. Kuczera, F. T. K. Lau,
C. Mattos, S. Michnick, T. Ngo, D. T. Nguyen, B. Prodhom,
W. E. Reiher, B. Roux, M. Schlenkrich, J. C. Smith, R. Stote,
J. Straub, M. Watanabe, J. Wiórkiewicz-Kuczera, D. Yin and
M. Karplus, J. Phys. Chem. B, 1998, 102, 3586–3616.
13 K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu,
S. Zhong, J. Shim, E. Darian, O. Guvench, P. Lopes,
I. Vorobyov and A. D. Mackerell Jr., J. Comput. Chem., 2010,
31, 671–690.
14 R. B. Best, X. Zhu, J. Shim, P. E. M. Lopes, J. Mittal, M. Feig
and A. D. MacKerell, J. Chem. Theory Comput., 2012, 8,
3257–3273.
This journal is © The Royal Society of Chemistry 2019
View Article Online
Paper
15 O. Guvench, S. S. Mallajosyula, E. P. Raman, E. Hatcher,
K. Vanommeslaeghe, T. J. Foster, F. W. Jamison and
A. D. MacKerell, J. Chem. Theory Comput., 2011, 7, 3162–
3180.
16 S. Nosé, J. Chem. Phys., 1984, 81, 511–519.
17 G. J. Martyna, D. J. Tobias and M. L. Klein, J. Chem. Phys.,
1994, 101, 4177–4189.
18 S. E. Feller, Y. Zhang, R. W. Pastor and B. R. Brooks,
J. Chem. Phys., 1995, 103, 4613–4621.
19 M. Karplus, J. Comput. Chem., 1983, 4, 187–217.
20 B. R. Brooks, C. L. Brooks, A. D. Mackerell, L. Nilsson,
R. J. Petrella, B. Roux, Y. Won, G. Archontis, C. Bartels,
S. Boresch, A. Caflisch, L. Caves, Q. Cui, A. R. Dinner,
M. Feig, S. Fischer, J. Gao, M. Hodoscek, W. Im,
K. Kuczera, T. Lazaridis, J. Ma, V. Ovchinnikov, E. Paci,
R. W. Pastor, C. B. Post, J. Z. Pu, M. Schaefer, B. Tidor,
R. M. Venable, H. L. Woodcock, X. Wu, W. Yang,
D. M. York and M. Karplus, J. Comput. Chem., 2009, 30,
1545–1614.
21 Y. Shao, L. F. Molnar, Y. Jung, J. Kussmann, C. Ochsenfeld,
S. T. Brown, A. T. B. Gilbert, L. V. Slipchenko,
S. V. Levchenko, D. P. O’Neill, R. A. DiStasio Jr.,
R. C. Lochan, T. Wang, G. J. O. Beran, N. A. Besley,
J. M. Herbert, C. Yeh Lin, T. Van Voorhis, S. Hung Chien,
A. Sodt, R. P. Steele, V. A. Rassolov, P. E. Maslen,
P. P. Korambath, R. D. Adamson, B. Austin, J. Baker,
E. F. C. Byrd, H. Dachsel, R. J. Doerksen, A. Dreuw,
B. D. Dunietz, A. D. Dutoi, T. R. Furlani, S. R. Gwaltney,
A. Heyden, S. Hirata, C.-P. Hsu, G. Kedziora,
R. Z. Khalliulin, P. Klunzinger, A. M. Lee, M. S. Lee,
W. Liang, I. Lotan, N. Nair, B. Peters, E. I. Proynov,
P. A. Pieniazek, Y. Min Rhee, J. Ritchie, E. Rosta, C. David
Sherrill, A. C. Simmonett, J. E. Subotnik, H. Lee Woodcock
III, W. Zhang, A. T. Bell, A. K. Chakraborty, D. M. Chipman,
F. J. Keil, A. Warshel, W. J. Hehre, H. F. Schaefer III,
J. Kong, A. I. Krylov, P. M. W. Gill and M. Head-Gordon,
Phys. Chem. Chem. Phys., 2006, 8, 3172–3191.
22 A. I. Krylov and P. M. W. Gill, Wiley Interdiscip. Rev.:
Comput. Mol. Sci., 2013, 3, 317–326.
23 Y. H. Shao, Z. T. Gan, E. Epifanovsky, A. T. B. Gilbert,
M. Wormit, J. Kussmann, A. W. Lange, A. Behn, J. Deng,
X. T. Feng, D. Ghosh, M. Goldey, P. R. Horn,
L. D. Jacobson, I. Kaliman, R. Z. Khaliullin, T. Kus,
A. Landau, J. Liu, E. I. Proynov, Y. M. Rhee, R. M. Richard,
M. A. Rohrdanz, R. P. Steele, E. J. Sundstrom,
H. L. Woodcock, P. M. Zimmerman, D. Zuev, B. Albrecht,
E. Alguire, B. Austin, G. J. O. Beran, Y. A. Bernard,
E. Berquist, K. Brandhorst, K. B. Bravaya, S. T. Brown,
D. Casanova, C. M. Chang, Y. Q. Chen, S. H. Chien,
K. D. Closser, D. L. Crittenden, M. Diedenhofen,
R. A. DiStasio, H. Do, A. D. Dutoi, R. G. Edgar, S. Fatehi,
L. Fusti-Molnar, A. Ghysels, A. Golubeva-Zadorozhnaya,
J. Gomes, M. W. D. Hanson-Heine, P. H. P. Harbach,
A. W. Hauser, E. G. Hohenstein, Z. C. Holden, T. C. Jagau,
H. J. Ji, B. Kaduk, K. Khistyaev, J. Kim, J. Kim, R. A. King,
P. Klunzinger, D. Kosenkov, T. Kowalczyk, C. M. Krauter,
Org. Biomol. Chem., 2019, 17, 6269–6276 | 6275

Paper
K. U. Lao, A. D. Laurent, K. V. Lawler, S. V. Levchenko,
C. Y. Lin, F. Liu, E. Livshits, R. C. Lochan, A. Luenser,
P. Manohar, S. F. Manzer, S. P. Mao, N. Mardirossian,
A. V. Marenich, S. A. Maurer, N. J. Mayhall,
E. Neuscamman, C. M. Oana, R. Olivares-Amaya,
D. P. O’Neill, J. A. Parkhill, T. M. Perrine, R. Peverati,
A. Prociuk, D. R. Rehn, E. Rosta, N. J. Russ, S. M. Sharada,
S. Sharma, D. W. Small, A. Sodt, T. Stein, D. Stuck, Y. C. Su,
A. J. W. Thom, T. Tsuchimochi, V. Vanovschi, L. Vogt,
O. Vydrov, T. Wang, M. A. Watson, J. Wenzel, A. White,
Published on 05 June 2019. Downloaded by KEAN UNIVERSITY on 7/17/2019 8:39:29 PM.
C. F. Williams, J. Yang, S. Yeganeh, S. R. Yost, Z. Q. You,
I. Y. Zhang, X. Zhang, Y. Zhao, B. R. Brooks, G. K. L. Chan,
D. M. Chipman, C. J. Cramer, W. A. Goddard,
M. S. Gordon, W. J. Hehre, A. Klamt, H. F. Schaefer,
M. W. Schmidt, C. D. Sherrill, D. G. Truhlar, A. Warshel,
X. Xu, A. Aspuru-Guzik, R. Baer, A. T. Bell, N. A. Besley,
J. D. Chai, A. Dreuw, B. D. Dunietz, T. R. Furlani,
S. R. Gwaltney, C. P. Hsu, Y. S. Jung, J. Kong,
6276 | Org. Biomol. Chem., 2019, 17, 6269–6276
View Article Online
Organic & Biomolecular Chemistry
D. S. Lambrecht, W. Z. Liang, C. Ochsenfeld,
V. A. Rassolov, L. V. Slipchenko, J. E. Subotnik, T. Van
Voorhis, J. M. Herbert, A. I. Krylov, P. M. W. Gill and
M. Head-Gordon, Mol. Phys., 2015, 113, 184–215.
24 A. T. Pereira, A. J. M. Ribeiro, P. A. Fernandes and
M. J. Ramos, Int. J. Quantum Chem., 2017, 117, e25409.
25 F. Mendoza, J. M. Lluch and L. Masgrau, Org. Biomol.
Chem., 2017, 15, 9095–9107.
26 A. R. Calixto, M. J. Ramos and P. A. Fernandes, J. Chem.
Theory Comput., 2017, 13, 5486–5495.
27 S. Fischer and M. Karplus, Chem. Phys. Lett., 1992, 194,
252–261.
28 N. Mardirossian and M. Head-Gordon, Phys. Chem. Chem.
Phys., 2014, 16, 9904–9924.
29 S. Dasgupta and J. M. Herbert, J. Comput. Chem., 2017, 38,
869–882.
30 D. Santos-Martins, A. R. Calixto, P. A. Fernandes and
M. J. Ramos, ACS Catal., 2018, 8, 4055–4063.
This journal is © The Royal Society of Chemistry 2019