QM/MM Molecular Dynamics Investigations of the Substrate Binding of Leucotriene A4 Hydrolase: Implication for the Catalytic Mechanism

LTA4H is a monozinc bifunctional enzyme which exhibits both aminopeptidase and epoxide hydrolase activities. Its dual functions in anti- and pro-inflammatory roles have attracted wide attention to the inhibitor design. In this work, we tried to construct Michaelis complexes of LTA4H with both a native peptide substrate and LTA4 molecule using combined quantum mechanics and molecular mechanics molecular dynamics simulations. First of all, the zinc ion is coordinated by H295, H299, and E318. For its aminopeptidase activity, similar to conventional peptidases, the fourth ligand to the zinc ion is suggested to be an active site water, which is further hydrogen bonded with a downstream glutamic acid, E296. For the epoxide hydrolase activity, the fourth ligand to the zinc ion is found to be an epoxy oxygen atom. The potential of mean force calculation indicates about an 8.5 kcal/mol activation barrier height for the ring-opening reaction, which will generate a metastable carbenium intermediate. Subsequent frontier molecular orbital analyses suggest that the next step would be the nucleophilic attacking reaction at the C12 atom by a water molecule activated by D375. Our simulations also analyzed functions of several important residues like R563, K565, E271, Y383, and Y378 in the binding of peptide and LTA4

Catalytic Mechanism of Hyaluronate Lyase from <i>Spectrococcus pneumonia</i>: Quantum Mechanical/Molecular Mechanical and Density Functional Theory Studies

Hyaluronate lyase from <i>Spectrococcus pneumonia</i> can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. The major functions of hyaluronan are to regulate water balance and osmotic pressure and act as an ion-exchange resin. It has been suggested in our previous molecular dynamics simulation that the binding of the substrate molecule could lead to the ionization of Y408 and protonation of H399. Followed by our recent molecular dynamics simulation of the enzyme–substrate complex, a unified proton abstraction and donation mechanism for this enzyme can be established using a combined quantum mechanical and molecular mechanical approach and density functional theory method. Y408 is shown to serve as the general base in the proton abstraction, while general acid is the next proton donation step. Overall, this reaction can be classified into <i>syn</i>-elimination reaction mechanism. The neutralization effects of C5 carboxylate group by several polar residues such as N349 and H399 were also examined. Finally, in combination of our previous molecular dynamics simulations, a complete catalytic cycle for the degradation of hyaluronan tetrasaccharide catalyzed by the hyaluronate lyase from <i>Spectrococcus pneumonia</i> is proposed

New Delhi Metallo-β-Lactamase I: Substrate Binding and Catalytic Mechanism

Metallo-β-lactamases can hydrolyze and deactivate lactam-containing antibiotics, which is the major mechanism for causing drug resistance in the treatment of bacterial infections. This has become a global concern because of the lack of clinically approved inhibitors so far. The emergence of New Delhi metallo-β-lactamase I (NDM-1) makes the situation even more serious. In this work, first, the structure of NDM-1 in complex with the inhibitor molecule l-captopril is investigated by both density functional theory (DFT) and hybrid quantum mechanical/molecular mechanical (QM/MM) methods, and the theoretical results are in good agreement with the X-ray structure. The Michaelis structure with an antibiotic compound (ampicillin) bound in the active site is constructed from a recent X-ray structure of the NDM-1 enzyme with hydrolyzed ampicillin. It is further simulated using a QM/MM molecular dynamics method. One of the interesting binding features of ampicillin in the NDM-1 active site is that the conserved C3 carboxylate group is not ligated with Zn2 but rather is only hydrogen-bonded with N220 and K211. A bridging hydroxide ion is suggested to connect two zinc cofactors. This hydroxide ion is also hydrogen-bonded with D124. Subsequent reaction path calculations indicate that the initial step of lactam ring-opening occurs through a concerted step in which the cleavage of the C–N bond and the transfer of the hydrogen bond to D124 are nearly concerted. The ligand bond between Zn2 and the C3 carboxylate group forms after the first step of nucleophilic addition. The calculated activation energy barrier height is about 19.4 kcal/mol for the hydrolysis of ampicillin, which can be compared with the experimental value of 15.8 kcal/mol derived from <i>k</i>_cat = 15 s^–1. The overall mechanism is finally confirmed by a subsequent DFT study of a truncated active-site model

Catalytic Mechanism of Angiotensin-Converting Enzyme and Effects of the Chloride Ion

The angiotensin-converting enzyme (ACE) exhibits critical functions in the conversion of angiotensin I to angiotensin II and the degradation of bradykinin and other vasoactive peptides. As a result, the ACE inhibition has become a promising approach in the treatment of hypertension, heart failure, and diabetic nephropathy. Extending our recent molecular dynamics simulation of the testis ACE in complex with a bona fide substrate molecule, hippuryl-histidyl-leucine, we presented here a detailed investigation of the hydrolytic process and possible influences of the chloride ion on the reaction using a combined quantum mechanical and molecule mechanical method. Similar to carboxypeptidase A and thermolysin, the promoted water mechanism is established for the catalysis of ACE. The E384 residue was found to have the dual function of a general base for activating the water nucleophile and a general acid for facilitating the cleavage of amide C–N bond. Consistent with experimental observations, the chloride ion at the second binding position is found to accelerate the reaction rate presumably due to the long-range electrostatic interactions but has little influence on the overall substrate binding characteristics

Initial Events in the Degradation of Hyaluronan Catalyzed by Hyaluronate Lyase from Spectrococcus pneumoniae: QM/MM Simulation

Hyaluronate lyase from Spectrococcus pneumonia can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. The major functions of hyaluronan are to regulate water balance and osmotic pressure and act as an ion-exchange resin. In this work, we focus on the prerequisite issue of the enzymatic reaction, i.e., the initial reactive conformer. Based on the quantum mechanical and molecular mechanical molecular dynamic simulations and free energy profiles, a near attack conformer was obtained for the degradation of hyaluronan catalyzed by the hyaluronate lyase. Along with the substrate binding, the phenylhydroxyl hydrogen atom of Tyr408 will transfer to nearby His399 via a near barrierless transition state, which results in a negatively charged Tyr408 and positively charged His399. The Tyr408, rather than the previously proposed His399, was suggested to act as the general base for the subsequent β-elimination reaction. The His399 was suggested to have the function of neutralizing the C5-carboxyl group

Quantum Mechanical/Molecular Mechanical Elucidation of the Catalytic Mechanism of Leukotriene A4 Hydrolase as an Epoxidase

Leukotriene A4 hydrolase (LTA4H) functions as a mono-zinc bifunctional enzyme with aminopeptidase and epoxidase activities. While the aminopeptidase mechanism is well understood, the epoxidase mechanism remains less clear. In continuation of our prior research, we undertook an in-depth exploration of the LTA4H catalytic role as an epoxidase, employing a combined SCC-DFTB/CHARMM method. In the current work, we found that the conversion of LTA4 to leukotriene B4 (LTB4) involves three successive steps: epoxy ring opening (RO), nucleophilic attack (NA), and proton transfer (PT) reactions at the epoxy oxygen atom. Among these steps, the RO and NA stages constitute the potential rate-limiting step within the entire epoxidase mechanism. Notably, the NA step implicates D375 as the general base catalyst, while the PT step engages protonated E271 as the general acid catalyst. Additionally, we delved into the mechanism behind the formation of the isomer product, Δ6-trans-Δ8-cis-LTB4. Our findings debunked the feasibility of a direct LTB4 to iso-LTB4 conversion. Instead, we highlight the possibility of isomerization from LTA4 to its isomeric conjugate (iso-LTA4), showing comparable energy barriers of 5.1 and 5.5 kcal/mol in aqueous and enzymatic environments, respectively. The ensuing dynamics of iso-LTA4 hydrolysis subsequently yield iso-LTB4 via a mechanism akin to LTA4 hydrolysis, albeit with a heightened barrier. Our computations firmly support the notion that substrate isomerization exclusively takes place prior to or during the initial substrate-binding phase, while LTA4 remains the dominant conformer. Notably, our simulations suggest that irrespective of the active site’s constrained L-shape, isomerization from LTA4 to its isomeric conjugate remains plausible. The mechanistic insights garnered from our simulations furnish a valuable understanding of LTA4H’s role as an epoxidase, thereby facilitating potential advancements in inhibitor design

“Amide Resonance” in the Catalysis of 1,2-α‑l‑Fucosidase from Bifidobacterium bifidum

Bifidobacterium is a genus of Gram-positive bacteria, which is important in the absorption of nourishment from the human milk oligosaccharides (HMO). We present here the detailed simulation of the enzymatic hydrolysis of 2′-fucosyllactose catalyzed by 1,2-α-l-fucosidase from Bifidobacterium bifidum using the combined quantum mechanical and molecular mechanical approach. Molecular dynamics simulations and free energy profiles support that the overall reaction is a stepwise mechanism. The first step is the proton transfer from N423 to D766, and the second step involves the hydrolysis reaction via the inversion mechanism catalyzed by the amide group of N423. Assisted by D766, N423 serves as the general base to activate the water molecule to attack the anomeric carbon center. E566 is the general acid to facilitate the cleavage of glycosidic bond between l-fucose and galactose units. The intrinsic resonance structure for the side chain amide group of the asparagine residue is shown to be the origin to the catalytic activity, which is also confirmed by the mutagenesis simulation of N423G

Surface Structure of Hydroxyapatite from Simulated Annealing Molecular Dynamics Simulations

The surface structure of hydroxyapatite (HAP) is crucial for its bioactivity. Using a molecular dynamics simulated annealing method, we studied the structure and its variation with annealing temperature of the HAP (100) surface. In contrast to the commonly used HAP surface model, which is sliced from HAP crystal and then relaxed at 0 K with first-principles or force-field calculations, a new surface structure with gradual changes from ordered inside to disordered on the surface was revealed. The disordering is dependent on the annealing temperature, <i>T</i>_max. When <i>T</i>_max increases up to the melting point, which was usually adopted in experiments, the disordering increases, as reflected by its radial distribution functions, structural factors, and atomic coordination numbers. The disordering of annealed structures does not show significant changes when <i>T</i>_max is above the melting point. The thickness of disordered layers is about 10 Å. The surface energy of the annealed structures at high temperature is significantly less than that of the crystal structure relaxed at room temperature. A three-layer model of interior, middle, and surface was then proposed to describe the surface structure of HAP. The interior layer retains the atomic configurations in crystal. The middle layer has its atoms moved and its groups rotated about their original locations. In the surface layer, the atomic arrangements are totally different from those in crystal. In particular for the hydroxyl groups, they move outward and cover the Ca²⁺ ions, leaving holes occupied by the phosphate groups. Our study suggested a new model with disordered surface structures for studying the interaction of HAP-based biomaterials with other molecules

Amine-Ligated Approach for the Synthesis of Extra-Large-Pore Zinc Phosphites with qtz‑h and bnn Topologies

Presented here are two open-framework zinc phosphites, namely, Zn­(dabco)_0.5(HPO₃) (SCU-18) and Zn₄(Hdabco)₂(CH₃COO)₂(HPO₃)₄ (SCU-20), where dabco = 1,4-diazabicyclo[2.2.2]­octane. SCU-18 features a rare 3-connected inorganic skeleton with a chiral qtz-h topology. It contains 18-membered-ring (18 MR) channels displaying porosity and second-harmonic-generation response. SCU-20 has a bnn topology containing large 20 MR channels that shows a strong blue emission as a result of excitation at 375 nm

Amine-Ligated Approach for the Synthesis of Extra-Large-Pore Zinc Phosphites with qtz‑h and bnn Topologies

Presented here are two open-framework zinc phosphites, namely, Zn­(dabco)_0.5(HPO₃) (SCU-18) and Zn₄(Hdabco)₂(CH₃COO)₂(HPO₃)₄ (SCU-20), where dabco = 1,4-diazabicyclo[2.2.2]­octane. SCU-18 features a rare 3-connected inorganic skeleton with a chiral qtz-h topology. It contains 18-membered-ring (18 MR) channels displaying porosity and second-harmonic-generation response. SCU-20 has a bnn topology containing large 20 MR channels that shows a strong blue emission as a result of excitation at 375 nm