10 research outputs found
Molecular Dynamics Simulations Elucidate Conformational Dynamics Responsible for the Cyclization Reaction in TEAS
The Mg-dependent 5-epi-aristolochene
synthase from <i>Nicotiana tabacum</i> (called TEAS) could
catalyze the linear farnesyl pyrophosphate (FPP) substrate to form
bicyclic hydrocarbon 5-epi-aristolochene. The cyclization reaction
mechanism of TEAS was proposed based on static crystal structures
and quantum chemistry calculations in a few previous studies, but
substrate FPP binding kinetics and protein conformational dynamics
responsible for the enzymatic catalysis are still unclear. Herein,
by elaborative and extensive molecular dynamics simulations, the loop
conformation change and several crucial residues promoting the cyclization
reaction in TEAS are elucidated. It is found that the unusual noncatalytic
NH<sub>2</sub>-terminal domain is essential to stabilize Helix-K and
the adjoining J-K loop of the catalytic COOH-terminal domain. It is
also illuminated that the induce-fit J-K/A-C loop dynamics is triggered
by Y527 and the optimum substrate binding mode in a āU-shapeā
conformation. The U-shaped ligand binding pose is maintained well
with the cooperative interaction of the three Mg<sup>2+</sup>-containing
coordination shell and conserved residue W273. Furthermore, the conserved
Arg residue pair R264/R266 and aromatic residue pair Y527/W273, whose
spatial orientations are also crucial to promote the closure of the
active site to a hydrophobic pocket, as well as to form Ļ-stacking
interactions with the ligand, would facilitate the carbocation migration
and electrophilic attack involving the catalytic reaction. Our investigation
more convincingly proves the greater roles of the protein local conformational
dynamics than do hints from the static crystal structure observations.
Thus, these findings can act as a guide to new protein engineering
strategies on diversifying the sesquiterpene products for drug discovery
Biosynthesis of Spinosyn A: A [4 + 2] or [6 + 4] Cycloaddition?
SpnF,
one of the DielsāAlderases, produces spinosyn A, and
previous work demonstrated that its sole function is to catalyze the [4 + 2] cycloaddition (Fage, C. D.; et al. Nat. Chem. Biol. 2015, 11, 256ā258). Furthermore, the potential existence of a [6 +
4] cycloaddition bifurcation from previous theoretical
calculations on the nonenzyme model (Patel, A.; et al. J.
Am. Chem. Soc. 2016, 138, 3631ā3634) shows that the
exact mechanism of SpnF becomes even more interesting as well as now
being controversial. In the present work, QMĀ(DFT)/MM MD simulations
on the full enzyme model revealed three significant residues that
collaborate with other residues to control the direction of the cycloaddition,
namely, Tyr23, Thr196, and Trp256. These residues force the substrate
into a reactive conformation that causes the cycloaddition reaction
to proceed through a [4 + 2] pathway
instead of the [6 + 4] one. The mechanistic
insights deciphered here are fundamentally important for the rational
design of DielsāAlderases and biomimetic syntheses
A Comprehensive Understanding of Enzymatic Catalysis by Hydroxynitrile Lyases with <i>S</i> Stereoselectivity from the Ī±/Ī²-Hydrolase Superfamily: Revised Role of the Active-Site Lysine and Kinetic Behavior of Substrate Delivery and Sequential Product Release
The highly homologous hydroxynitrile
lyases from Manihot esculent (<i>Me</i>HNL) and Hevea brasiliensis (<i>Hb</i>HNL) both belong to the Ī±/Ī²-hydrolase
superfamily, and they convert cyanohydrins into the corresponding
ketone (aldehyde) and hydrocyanic acid, which is important for biosynthesis
for carbonācarbon formation. On the basis of extensive MM and
ab initio QM/MM MD simulations, one-dimensional and two-dimensional
free energy profiles on the whole enzymatic catalysis by <i>Me</i>HNL have been explored, and the effects of key residues around the
channel on the delivery of substrate and product have been discussed.
The residue Trp128 plays an important gate-switching role to manipulate
the substrate access to the active site and product release. In particular,
the release of acetone and HCN has been first detected to follow a
stepwise mechanism. The release of HCN is quite facile, while the
escape of acetone experiences a barrier of ā¼10 kcal/mol. The
chemical reaction is an endergonic process with a free energy barrier
of ā¼17.1 kcal/mol, which dominates the entire enzymatic efficiency.
Such energy costs can be compensated by the remarkable energy release
during the initial substrate binding. Here the carbonācarbon
cleavage is the rate-determining step, which differs from that of <i>Hb</i>HNL. The protonation state of Lys237 plays an important
role in carbonācarbon bond cleavage by restoring the Ser80Ala
mutant system to the wild system, which explains the discrepancy between <i>Me</i>HNL and <i>Hb</i>HNL at the molecular or atomic
scale. The present results provide a basis for understanding the similarity
and difference in the enzymatic catalysis by <i>Me</i>HNL
and <i>Hb</i>HNL
Protonation-Dependent Diphosphate Cleavage in FPP Cyclases and Synthases
The cleavage of the
magnesium-assisted diphosphate group (the PPi
group) is one significant and prevalent rate-limiting step triggering
the enzyme catalysis synthesis of terpenoid natural products. However,
the PPi cleavage procedure has been rarely studied in most theoretical
research of the terpenoid biosynthetic mechanism. In this work, QMĀ(DFT)/MM
MD simulations were employed to illuminate the detailed PPi cleavage
mechanism in three different enzyme systems (ATAS, TEAS, and FPPS).
We found that the most rational protonation state of the PPi group
is highly dependent on the Mg<sup>2+</sup> coordination modes and
the enzyme classes. The deprotonation of PPi is favorable for triggering
the catalysis reaction in ATAS, while monoprotonation in FPPS and
biprotonation in TEAS are advantageous. As a result, similar PPi cleavage
occurs by means of nucleophilic substitution reactions in TEAS/FPPS/ATAS
but presents an S<sub>N</sub>1, S<sub>N</sub>2, and borderline mechanism,
respectively. Finally, the alternative functions of PPi protonation
and Mg<sup>2+</sup> coordination modes are discussed
QM/MM and MM MD Simulations on the Pyrimidine-Specific Nucleoside Hydrolase: A Comprehensive Understanding of Enzymatic Hydrolysis of Uridine
The pyrimidine-specific
nucleoside hydrolase Yeik (CU-NH) from Escherichia
coli cleaves the N-glycosidic bond of uridine and
cytidine with a 10<sup>2</sup>ā10<sup>4</sup>-fold faster rate
than that of purine nucleoside substrates, such as inosine. Such a
remarkable substrate specificity and the plausible hydrolytic mechanisms
of uridine have been explored by using QM/MM and MM MD simulations.
The present calculations show that the relatively stronger hydrogen-bond
interactions between uridine and the active-site residues Gln227 and
Tyr231 in CU-NH play an important role in enhancing the substrate
binding and thus promoting the N-glycosidic bond cleavage, in comparison
with inosine. The estimated energy barrier of 30 kcal/mol for the
hydrolysis of inosine is much higher than 22 kcal/mol for uridine.
Extensive MM MD simulations on the transportation of substrates to
the active site of CU-NH indicate that the uridine binding is exothermic
by ā¼23 kcal/mol, more remarkable than inosine (ā¼12 kcal/mol).
All of these arise from the noncovalent interactions between the substrate
and the active site featured in CU-NH, which account for the substrate
specificity. Quite differing from other nucleoside hydrolases, here
the enzymatic N-glycosidic bond cleavage of uridine is less influenced
by its protonation
Concerted Cyclization of Lanosterol CāRing and DāRing Under Human Oxidosqualene Cyclase Catalysis: An ab Initio QM/MM MD Study
Human oxidosqualene cyclase (OSC)
is one key enzyme in the biosynthesis
of cholesterol. It can catalyze the linear-chain 2,3-oxidosqualene
to form lanosterol, the tetracyclic (6ā6ā6ā5
members for AāBāCāD rings) cholesterol precursor.
It also has been treated as a novel antihyperlipidemia target. In
addition, the structural diversity of cyclic terpenes in plants originates
from the cyclization of 2,3-oxidosqualene. The enzyme catalytic mechanism
is considered to be one of the most complicated ones in nature, and
there are a lot of controversies about the mechanism in the past half
a century. Herein, state-of-the-art ab initio QM/MM MD simulations
are employed to investigate the detailed cyclization mechanism of
C-ring and D-ring formation. Our study reveals that the C and D rings
are formed near-synchronously from a stable ā6ā6ā5ā
ring intermediate. Interestingly, the transition state of this concerted
reaction presents a ā6ā6-6ā structure motif,
while this unstable ā6ā6-6ā structure in our
simulations is thought to be a stable intermediate state in most previous
hypothetical mechanisms. Furthermore, as the tailed side chain of
2,3-oxidosqualene shows a Ī² conformation while it is Ī±
conformation in lanosterol, finally, it is observed that the rotatable
ātailā chain prefers to transfer Ī² conformation
to Ī± conformation at the ā6ā6ā5ā
intermediate state
Mechanistic Insights into the Rate-Limiting Step in Purine-Specific Nucleoside Hydrolase
A full enzymatic catalysis cycle
in the inosineāadenosineāguanosine
specific nucleoside hydrolase (IAG-NH) was assumed to be comprised
of four steps: substrate binding, chemical reaction, base release,
and ribose release. Nevertheless, the mechanistic details for the
rate-limiting step of the entire enzymatic reaction are still unknown,
even though the ribose release was likely to be the most difficult
stage. Based on state-of-the-art quantum mechanics and molecular mechanics
(QM/MM) molecular dynamics (MD) simulations, the ribose release process
can be divided into two steps: āribose dissociationā
and āribose releaseā. The āribose dissociationā
includes ācleavageā and āexchangeā stages,
in which a metastable 6-fold intermediate will recover to an 8-fold
coordination shell of Ca<sup>2+</sup> as observed in <i>apo</i>- IAG-NH. Extensive random acceleration molecular dynamics and MD
simulations have been employed to verify plausible release channels,
and the estimated barrier for the rate-determining step of the entire
reaction is 13.0 kcal/mol, which is comparable to the experimental
value of 16.7 kcal/mol. Moreover, the gating mechanism arising from
loop1 and loop2, as well as key residues around the active pocket,
has been found to play an important role in manipulating the ribose
release
Computational Design of a Time-Dependent Histone Deacetylase 2 Selective Inhibitor
Development of isoform-selective
histone deacetylase (HDAC) inhibitors is of great biological and medical
interest. Among 11 zinc-dependent HDAC isoforms, it is particularly
challenging to achieve isoform inhibition selectivity between HDAC1
and HDAC2 due to their very high structural similarities. In this
work, by developing and applying a novel de novo reaction-mechanism-based
inhibitor design strategy to exploit the reactivity difference, we
have discovered the first HDAC2-selective inhibitor, Ī²-hydroxymethyl
chalcone. Our bioassay experiments show that this new compound has
a unique time-dependent selective inhibition on HDAC2, leading to
about 20-fold isoform-selectivity against HDAC1. Furthermore, our
ab initio QM/MM molecular dynamics simulations, a state-of-the-art
approach to study reactions in biological systems, have elucidated
how the Ī²-hydroxymethyl chalcone can achieve the distinct time-dependent
inhibition toward HDAC2
Molecular Dynamics-Based Virtual Screening: Accelerating the Drug Discovery Process by High-Performance Computing
High-performance computing (HPC)
has become a state strategic technology
in a number of countries. One hypothesis is that HPC can accelerate
biopharmaceutical innovation. Our experimental data demonstrate that
HPC can significantly accelerate biopharmaceutical innovation by employing
molecular dynamics-based virtual screening (MDVS). Without using HPC,
MDVS for a 10K compound library with tens of nanoseconds of MD simulations
requires years of computer time. In contrast, a state of the art HPC
can be 600 times faster than an eight-core PC server is in screening
a typical drug target (which contains about 40K atoms). Also, careful
design of the GPU/CPU architecture can reduce the HPC costs. However,
the communication cost of parallel computing is a bottleneck that
acts as the main limit of further virtual screening improvements for
drug innovations
Molecular Dynamics-Based Virtual Screening: Accelerating the Drug Discovery Process by High-Performance Computing
High-performance computing (HPC)
has become a state strategic technology
in a number of countries. One hypothesis is that HPC can accelerate
biopharmaceutical innovation. Our experimental data demonstrate that
HPC can significantly accelerate biopharmaceutical innovation by employing
molecular dynamics-based virtual screening (MDVS). Without using HPC,
MDVS for a 10K compound library with tens of nanoseconds of MD simulations
requires years of computer time. In contrast, a state of the art HPC
can be 600 times faster than an eight-core PC server is in screening
a typical drug target (which contains about 40K atoms). Also, careful
design of the GPU/CPU architecture can reduce the HPC costs. However,
the communication cost of parallel computing is a bottleneck that
acts as the main limit of further virtual screening improvements for
drug innovations