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 Asp₁₂₄. 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^–] 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^– 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 Tyr₁₅₀ deprotonated and both Lys₆₇ and Lys₃₁₅ residues being protonated is the most stable one, both thermodynamically and kinetically. However, the equilibrium structure at room temperature is a dynamic one, with Lys₃₁₅H_ζ delocalized between Tyr₁₅₀O_η and Lys₃₁₅N_ζ. 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 Tyr₁₅₀O_η and Lys₆₇N_ζ. 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 Lys₆₇, while Tyr₁₅₀ 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 Lys₆₇, while Tyr₁₅₀ 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 (Ser₆₄). 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 Tyr₁₅₀ deprotonated and both Lys₆₇ and Lys₃₁₅ residues being protonated is the most stable one, both thermodynamically and kinetically. However, the equilibrium structure at room temperature is a dynamic one, with Lys₃₁₅H_ζ delocalized between Tyr₁₅₀O_η and Lys₃₁₅N_ζ. 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 Tyr₁₅₀O_η and Lys₆₇N_ζ. 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 Lys₆₇, while Tyr₁₅₀ 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 Tyr₁₅₀ deprotonated and both Lys₆₇ and Lys₃₁₅ residues being protonated is the most stable one, both thermodynamically and kinetically. However, the equilibrium structure at room temperature is a dynamic one, with Lys₃₁₅H_ζ delocalized between Tyr₁₅₀O_η and Lys₃₁₅N_ζ. 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 Tyr₁₅₀O_η and Lys₆₇N_ζ. 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