16 research outputs found
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 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
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
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
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
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
Upregulation of p53 through induction of MDM2 degradation: improved potency through the introduction of an alkylketone sidechain on the anthraquinone core
Overexpression of ubiquitin ligase MDM2 causes depletion of the p53 tumour-suppressor and thus leads to cancer progression. In recent years, anthraquinone analogs have received significant attention due to their ability to downregulate MDM2, thereby promoting p53-induced apoptosis. Previously, we have developed potent anthraquinone compounds having the ability to upregulate p53 via inhibition of MDM2 in both cell culture and animal models of acute lymphocytic leukaemia. Earlier work was focussed on mechanistic work, pharmacological validation of this class of compounds in animal models, and mapping out structural space that allows for further modification and optimisation. Herein, we describe our work in optimising the substituents on the two phenol hydroxyl groups. It was found that the introduction of an alkylketone moiety led to a potent series of analogs with BW-AQ-350 being the most potent compound yet (IC50 = 0.19 ± 0.01 µM) which exerts cytotoxicity by inducing MDM2 degradation and p53 upregulation.</p