18 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
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
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
Mechanism of Assembling Isoprenoid Building Blocks 1. Elucidation of the Structural Motifs for Substrate Binding in Geranyl Pyrophosphate Synthase
Terpenes
(isoprenoids) represent the most functionally and structurally
diverse group of natural products. Terpenes are assembled from two
building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP or DPP), by prenyltransferases (PTSs). Geranyl pyrophosphate
synthase (GPPS) is the enzyme that assembles DPP and IPP in the first
step of chain elongation during isoprenoid biosynthesis. The mechanism
by which GPPS assembles the terpene precursor remains unknown; elucidating
this mechanism will help in development of new technology to generate
novel natural product-like scaffolds. With classic and QM/MM MD simulations,
an āopen-closedā conformation change of the catalytic
pocket was observed in the GPPS active site at its large subunit (LSU),
and a critical salt bridge between Asp91Ā(in loop 1) and Lys239Ā(in
loop 2) was identified. The salt bridge is responsible for opening
or closing the catalytic pocket. Meanwhile, the small subunit (SSU)
regulates the size and shape of the hydrophobic pocket to flexibly
host substrates with different shapes and sizes (DPP/GPP/FPP, C<sub>5</sub>/C<sub>10</sub>/C<sub>15</sub>). Further QM/MM MD simulations
were carried out to explore the binding modes for the different substrates
catalyzed by GPPS. Our simulations suggest that the key residues (Asp91,
Lys239, and Gln156) are good candidates for site-directed mutagenesis
and may help in protein engineering
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
Intrinsic Dynamics of the Binding Rail and Its Allosteric Effect in the Class I Histone Deacetylases
The development of novel isoform/class-selective
inhibitors is
still of great biological and medical significance to conquer the
continuously reported side effects for the histone deacetylase (HDAC)
drugs. The first potent HDAC allosteric inhibitor was discovered last
year, and this allosteric inhibitor design is thought to be a promising
strategy to overcome the current challenges in HDAC inhibitor design.
However, the detailed allosteric mechanism and its remote regulatory
effects on the catalytic/inhibitor activity of HDAC are still unclear.
In this work, on the basis of microsecond-time-scale all-atom molecular
dynamics (MD) simulations and picosecond-time-scale density functional
theory/molecular mechanics MD simulations on HDAC8, we propose that
the allostery is achieved by the intrinsic conformational flexibility
of the binding rail (constituted by a highly conserved XāD
residue dyad), which steers the loopāloop motion and creates
the diverse shapes of the allosteric sites in different HDAC isoforms.
Additionally, the rotatability of the binding rail is an inherent
structural feature that regulates the hydrophobicity of the linker
binding channel and thus further affects the HDAC enzyme inhibitory/catalytic
activity by utilizing the promiscuity of XāD dyad. Since the
plastic X residue is different among class I HDACs, these new findings
provide a deeper understanding of the allostery, which is guidable
for the design of new allosteric inhibitors toward the allosteric
site and structure modifications on the conventional inhibitors binding
into the active pocket by exploiting the intrinsic dynamic features
of the conserved XāD dyad
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
Intrinsic Dynamics of the Binding Rail and Its Allosteric Effect in the Class I Histone Deacetylases
The development of novel isoform/class-selective
inhibitors is
still of great biological and medical significance to conquer the
continuously reported side effects for the histone deacetylase (HDAC)
drugs. The first potent HDAC allosteric inhibitor was discovered last
year, and this allosteric inhibitor design is thought to be a promising
strategy to overcome the current challenges in HDAC inhibitor design.
However, the detailed allosteric mechanism and its remote regulatory
effects on the catalytic/inhibitor activity of HDAC are still unclear.
In this work, on the basis of microsecond-time-scale all-atom molecular
dynamics (MD) simulations and picosecond-time-scale density functional
theory/molecular mechanics MD simulations on HDAC8, we propose that
the allostery is achieved by the intrinsic conformational flexibility
of the binding rail (constituted by a highly conserved XāD
residue dyad), which steers the loopāloop motion and creates
the diverse shapes of the allosteric sites in different HDAC isoforms.
Additionally, the rotatability of the binding rail is an inherent
structural feature that regulates the hydrophobicity of the linker
binding channel and thus further affects the HDAC enzyme inhibitory/catalytic
activity by utilizing the promiscuity of XāD dyad. Since the
plastic X residue is different among class I HDACs, these new findings
provide a deeper understanding of the allostery, which is guidable
for the design of new allosteric inhibitors toward the allosteric
site and structure modifications on the conventional inhibitors binding
into the active pocket by exploiting the intrinsic dynamic features
of the conserved XāD dyad
StructureāFunction Analysis of the Conserved Tyrosine and Diverse ĻāStacking among Class I Histone Deacetylases: A QM (DFT)/MM MD Study
Discovery of the isoform-selective histone deacetylases
(HDACs) inhibitors is of great medical importance and still a challenge.
The comparison studies on the structureāfunction relationship
of the conserved residues, which are located in the linker binding
channel among class I HDACs (including 4 isoforms: HDAC1/2/3/8), have
been carried out by using <i>ab initio</i> QM/MM MD simulations,
a state-of-the-art approach to simulate metallo-enzymes. We found
that the conserved tyrosine (Y303/308/286/306 in HDAC1/2/3/8, respectively)
could modulate the zinc-inhibitor chelation among all class I HDACs
with different regulatory mechanisms. For HDAC1/2/3 selective-inhibitor
benzamide, the conserved tyrosine could modulate the coordinative
ability of the central atom (Zn<sup>2+</sup>), while for pan-inhibitor
SAHA, the conserved tyrosine could increase the chelating ability
of the ligand (SAHA). Moreover, it is first found that the conserved
tyrosine is correlated with the intertransformation of ĻāĻ
stacking styles (parallel shift vs T-shaped) by the aromatic ring
in benzamide and the two conserved phenylalanine residues of HDACs.
In addition, the catalytic roles of the conserved tyrosine in stabilizing
the transition state and intermediate are further revealed. These
findings provide useful molecular basis knowledge for further isoform-selective
inhibitor design among class I HDACs
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