17 research outputs found
Mechanism of Meropenem Hydrolysis by New Delhi Metallo β‑Lactamase
New Delhi metallo β-lactamase
(NDM-1) is a recent addition
to the metallo-β-lactamases family that is capable of hydrolyzing
most of the available antibiotics, including the new generation carbapenems.
Here, we report the mechanism of Meropenem hydrolysis catalyzed by
NDM-1 based on hybrid quantum-mechanical/molecular-mechanical metadynamics
simulations. Our work elicits the molecular details of the catalytic
mechanism and free energy profiles along the reaction pathway. We
identified the ring opening step involving the nucleophilic addition
of the bridging hydroxyl group on the β-lactam ring of the drug
as the rate-determining step. Subsequent protonation of β-lactam
nitrogen occurs from a bulk water molecule that diffuses into the
active site and is preferred over proton transfer from the bridging
hydroxyl group or from the protonated Asp<sub>124</sub>. The roles
of important active site residues of NDM-1 and change in the coordination
environment of Zn ions during the hydrolysis are also scrutinized
Can the Absence of Isotope Scrambling in the Wacker Oxidation of Allyl Alcohol Disprove Outer Sphere Hydroxypalladation?
The major controversy regarding the
mechanism of the Wacker oxidation
of alkenes at low [Cl<sup>–</sup>] concerns the mechanism of
nucleophilic attack of a solvent water molecule on alkene. Most of
the recent mechanistic studies on the Wacker oxidation of ethene have
reported that nucleophilic attack occurs by an outer sphere mechanism
and not by an inner sphere mechanism. One of the crucial experimental
findings in support of the inner sphere mechanism is that isotope
scrambling does not take place when deuterated ally alcohol was oxidized
under the standard Wacker conditions. In this work, we try to explain
these experimental results in the framework of the outer sphere mechanism.
We simulated the Wacker oxidation of allyl alcohol using ab initio
molecular dynamics (AIMD) techniques in order to probe the detailed
mechanism, free-energy profiles, and the rate-determining step. Our
simulations show that the mechanism of allyl alcohol oxidation follows
outer sphere hydroxypalladation, and the rate-determining step involves
Cl<sup>–</sup> ligand isomerization, contradicting the conclusions
from the isotope scrambling experiments. However, by carrying out
microkinetic modeling based on the free-energy barriers of the elementary
steps obtained from our AIMD simulations, we also observe no isotope
scrambling. This led us to determine the genesis of the observed absence
of isotope scrambling. Most importantly, here we demonstrate that
the absence of isotope scrambling is in fact consistent with the outer
sphere hydroxypalladation and cannot disprove it
Thermodynamic and Kinetic Stabilities of Active Site Protonation States of Class C β‑Lactamase
By employing computationally intensive molecular dynamics
simulations
using hybrid quantum–mechanical/molecular–mechanical
approach, we analyze here the kinetic and thermodynamic stabilities
of various active site protonation states of a fully solvated class
C β‑lactamase. We report the detailed mechanism of proton
transfer between catalytically important active site residues and
the associated free energy barriers. In the apoenzyme, significant
structural changes are associated with the proton transfer, and the
orientations of active site residues are distinctly different for
various protonation states. Among several propositions on the protonation
state of the apoprotein, we find that the one with Tyr<sub>150</sub> deprotonated and both Lys<sub>67</sub> and Lys<sub>315</sub> residues
being protonated is the most stable one, both thermodynamically and
kinetically. However, the equilibrium structure at room temperature
is a dynamic one, with Lys<sub>315</sub>H<sub>ζ</sub> delocalized
between Tyr<sub>150</sub>O<sub>η</sub> and Lys<sub>315</sub>N<sub>ζ</sub>. Of great importance, the kinetic and thermodynamic
stability of protonation states are significantly affected on noncovalently
complexing with cephalothin, an antibiotic molecule. The equilibrium
structure of the enzyme–substrate (precovalent) complex has
a dynamic protonation state where a proton shuttles frequently between
the Tyr<sub>150</sub>O<sub>η</sub> and Lys<sub>67</sub>N<sub>ζ</sub>. We examine here the genesis of the manifold change
in stability at the molecular level. The importance of our observations
toward understanding the reactivity of the enzyme is discussed and
experimental observations are rationalized
Mechanism of Acyl–Enzyme Complex Formation from the Henry–Michaelis Complex of Class C β‑Lactamases with β‑Lactam Antibiotics
Bacteria that cause most of the hospital-acquired
infections make
use of class C β-lactamase (CBL) among other enzymes to resist
a wide spectrum of modern antibiotics and pose a major public health
concern. Other than the general features, details of the defensive
mechanism by CBL, leading to the hydrolysis of drug molecules, remain
a matter of debate, in particular the identification of the general
base and role of the active site residues and substrate. In an attempt
to unravel the detailed molecular mechanism, we carried out extensive
hybrid quantum mechanical/molecular mechanical Car–Parrinello
molecular dynamics simulation of the reaction with the aid of the
metadynamics technique. On this basis, we report here the mechanism
of the formation of the acyl–enzyme complex from the Henry–Michaelis
complex formed by β-lactam antibiotics and CBL. We considered
two β-lactam antibiotics, namely, cephalothin and aztreonam,
belonging to two different subfamilies. A general mechanism for the
formation of a β-lactam antibiotic–CBL acyl–enzyme
complex is elicited, and the individual roles of the active site residues
and substrate are probed. The general base in the acylation step has
been identified as Lys<sub>67</sub>, while Tyr<sub>150</sub> aids
the protonation of the β-lactam nitrogen through either the
substrate carboxylate group or a water molecule
Mechanism of Acyl–Enzyme Complex Formation from the Henry–Michaelis Complex of Class C β‑Lactamases with β‑Lactam Antibiotics
Bacteria that cause most of the hospital-acquired
infections make
use of class C β-lactamase (CBL) among other enzymes to resist
a wide spectrum of modern antibiotics and pose a major public health
concern. Other than the general features, details of the defensive
mechanism by CBL, leading to the hydrolysis of drug molecules, remain
a matter of debate, in particular the identification of the general
base and role of the active site residues and substrate. In an attempt
to unravel the detailed molecular mechanism, we carried out extensive
hybrid quantum mechanical/molecular mechanical Car–Parrinello
molecular dynamics simulation of the reaction with the aid of the
metadynamics technique. On this basis, we report here the mechanism
of the formation of the acyl–enzyme complex from the Henry–Michaelis
complex formed by β-lactam antibiotics and CBL. We considered
two β-lactam antibiotics, namely, cephalothin and aztreonam,
belonging to two different subfamilies. A general mechanism for the
formation of a β-lactam antibiotic–CBL acyl–enzyme
complex is elicited, and the individual roles of the active site residues
and substrate are probed. The general base in the acylation step has
been identified as Lys<sub>67</sub>, while Tyr<sub>150</sub> aids
the protonation of the β-lactam nitrogen through either the
substrate carboxylate group or a water molecule
Deacylation Mechanism and Kinetics of Acyl–Enzyme Complex of Class C β‑Lactamase and Cephalothin
Understanding the molecular details
of antibiotic resistance by
the bacterial enzymes β-lactamases is vital for the development
of novel antibiotics and inhibitors. In this spirit, the detailed
mechanism of deacylation of the acyl–enzyme complex formed
by cephalothin and class C β-lactamase is investigated here
using hybrid quantum-mechanical/molecular-mechanical molecular dynamics
methods. The roles of various active-site residues and substrate in
the deacylation reaction are elucidated. We identify the base that
activates the hydrolyzing water molecule and the residue that protonates
the catalytic serine (Ser<sub>64</sub>). Conformational changes in
the active sites and proton transfers that potentiate the efficiency
of the deacylation reaction are presented. We have also characterized
the oxyanion holes and other H-bonding interactions that stabilize
the reaction intermediates. Together with the kinetic and mechanistic
details of the acylation reaction, we analyze the complete mechanism
and the overall kinetics of the drug hydrolysis. Finally, the apparent
rate-determining step in the drug hydrolysis is scrutinized
Thermodynamic and Kinetic Stabilities of Active Site Protonation States of Class C β‑Lactamase
By employing computationally intensive molecular dynamics
simulations
using hybrid quantum–mechanical/molecular–mechanical
approach, we analyze here the kinetic and thermodynamic stabilities
of various active site protonation states of a fully solvated class
C β‑lactamase. We report the detailed mechanism of proton
transfer between catalytically important active site residues and
the associated free energy barriers. In the apoenzyme, significant
structural changes are associated with the proton transfer, and the
orientations of active site residues are distinctly different for
various protonation states. Among several propositions on the protonation
state of the apoprotein, we find that the one with Tyr<sub>150</sub> deprotonated and both Lys<sub>67</sub> and Lys<sub>315</sub> residues
being protonated is the most stable one, both thermodynamically and
kinetically. However, the equilibrium structure at room temperature
is a dynamic one, with Lys<sub>315</sub>H<sub>ζ</sub> delocalized
between Tyr<sub>150</sub>O<sub>η</sub> and Lys<sub>315</sub>N<sub>ζ</sub>. Of great importance, the kinetic and thermodynamic
stability of protonation states are significantly affected on noncovalently
complexing with cephalothin, an antibiotic molecule. The equilibrium
structure of the enzyme–substrate (precovalent) complex has
a dynamic protonation state where a proton shuttles frequently between
the Tyr<sub>150</sub>O<sub>η</sub> and Lys<sub>67</sub>N<sub>ζ</sub>. We examine here the genesis of the manifold change
in stability at the molecular level. The importance of our observations
toward understanding the reactivity of the enzyme is discussed and
experimental observations are rationalized
Mechanism of Acyl–Enzyme Complex Formation from the Henry–Michaelis Complex of Class C β‑Lactamases with β‑Lactam Antibiotics
Bacteria that cause most of the hospital-acquired
infections make
use of class C β-lactamase (CBL) among other enzymes to resist
a wide spectrum of modern antibiotics and pose a major public health
concern. Other than the general features, details of the defensive
mechanism by CBL, leading to the hydrolysis of drug molecules, remain
a matter of debate, in particular the identification of the general
base and role of the active site residues and substrate. In an attempt
to unravel the detailed molecular mechanism, we carried out extensive
hybrid quantum mechanical/molecular mechanical Car–Parrinello
molecular dynamics simulation of the reaction with the aid of the
metadynamics technique. On this basis, we report here the mechanism
of the formation of the acyl–enzyme complex from the Henry–Michaelis
complex formed by β-lactam antibiotics and CBL. We considered
two β-lactam antibiotics, namely, cephalothin and aztreonam,
belonging to two different subfamilies. A general mechanism for the
formation of a β-lactam antibiotic–CBL acyl–enzyme
complex is elicited, and the individual roles of the active site residues
and substrate are probed. The general base in the acylation step has
been identified as Lys<sub>67</sub>, while Tyr<sub>150</sub> aids
the protonation of the β-lactam nitrogen through either the
substrate carboxylate group or a water molecule
Thermodynamic and Kinetic Stabilities of Active Site Protonation States of Class C β‑Lactamase
By employing computationally intensive molecular dynamics
simulations
using hybrid quantum–mechanical/molecular–mechanical
approach, we analyze here the kinetic and thermodynamic stabilities
of various active site protonation states of a fully solvated class
C β‑lactamase. We report the detailed mechanism of proton
transfer between catalytically important active site residues and
the associated free energy barriers. In the apoenzyme, significant
structural changes are associated with the proton transfer, and the
orientations of active site residues are distinctly different for
various protonation states. Among several propositions on the protonation
state of the apoprotein, we find that the one with Tyr<sub>150</sub> deprotonated and both Lys<sub>67</sub> and Lys<sub>315</sub> residues
being protonated is the most stable one, both thermodynamically and
kinetically. However, the equilibrium structure at room temperature
is a dynamic one, with Lys<sub>315</sub>H<sub>ζ</sub> delocalized
between Tyr<sub>150</sub>O<sub>η</sub> and Lys<sub>315</sub>N<sub>ζ</sub>. Of great importance, the kinetic and thermodynamic
stability of protonation states are significantly affected on noncovalently
complexing with cephalothin, an antibiotic molecule. The equilibrium
structure of the enzyme–substrate (precovalent) complex has
a dynamic protonation state where a proton shuttles frequently between
the Tyr<sub>150</sub>O<sub>η</sub> and Lys<sub>67</sub>N<sub>ζ</sub>. We examine here the genesis of the manifold change
in stability at the molecular level. The importance of our observations
toward understanding the reactivity of the enzyme is discussed and
experimental observations are rationalized
Presentation_1_Interfacing the Core-Shell or the Drude Polarizable Force Field With Car–Parrinello Molecular Dynamics for QM/MM Simulations.PDF
<p>We report a quantum mechanics/polarizable–molecular mechanics (QM/p–MM) potential based molecular dynamics (MD) technique where the core–shell (or the Drude) type polarizable MM force field is interfaced with the plane-wave density functional theory based QM force field which allows Car–Parrinello MD for the QM subsystem. In the QM/p-MM Lagrangian proposed here, the shell (or the Drude) MM variables are treated as extended degrees of freedom along with the Kohn–Sham (KS) orbitals describing the QM wavefunction. The shell and the KS orbital degrees of freedom are then adiabatically decoupled from the nuclear degrees of freedom. In this respect, we also present here the Nosé–Hoover Chain thermostat implementation for the dynamical subsystems. Our approach is then used to investigate the effect of MM polarization on the QM/MM results. Especially, the consequence of MM polarization on reaction free energy barriers, defect formation energy, and structural and dynamical properties are investigated. A low point charge polarizable potential (p–MZHB) for pure siliceous systems is also reported here.</p