Theoretical study of the full reaction mechanism of human soluble epoxide hydrolase

The complete reaction mechanism of soluble epoxide hydrolase (sEH) has been investigated by using the B3LYP density functional theory method. Epoxide hydrolases catalyze the conversion of epoxides to their corresponding vicinal diols. In our theoretical study, the sEH active site is represented by quantum-chemical models that are based on the X-ray crystal structure of human soluble epoxide hydrolase. The trans-substituted epoxide (1S,2S)-beta-methylstyrene oxide has been used as a substrate in the theoretical investigation of the sEH reaction mechanism. Both the alkylation and the hydrolytic half-reactions have been studied in detail. We present the energetics of the reaction mechanism as well as the optimized intermediates and transition-state structures. Full potential energy curves for the reactions involving nucleophilic attack at either the benzylic or the homo-benzylic carbon atom of (1S,2S)-beta-methylstyrene oxide have been computed. The regioselectivity of epoxide opening has been addressed for the two substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide.     

Reaction mechanism of soluble epoxide hydrolase: insights from molecular dynamics simulations

Molecular dynamics simulations have been performed to gain insights into the catalytic mechanism of the hydrolysis of epoxides to vicinal diols by soluble epoxide hydrolase (sEH). The binding of a substrate, 1S,2S-trans-methylstyrene oxide, was studied in two conformations in the active site of the enzyme. It was found that only one is likely to be found in the active enzyme. In the preferred conformation the phenyl group of the substrate is pi-sandwiched between two aromatic residues, Tyr381 and His523, whereas the other conformation is pi-stacked with only one aromatic residue, Trp334. Two simulations were carried out to 1 ns for each conformation to evaluate the protonation state of active site residue His523. It was found that a protonated histidine is essential for keeping the active site from being disrupted. Long time scale, 4 ns, molecular dynamics simulation was done for the structure with the most likely combination of binding conformation and protonation state of His523. Near Attack Conformers (NACs) are present 5.3% of the time and nucleophilic attack on either epoxide carbon atom, approximately 75% on C(1) and approximately 25% on C(2), is found. A maximum of one hydrogen bond between the epoxide oxygen and either of the active site tyrosines, Tyr465 and Tyr381, is present, in agreement with experimental mutagenesis results that reveal a slight loss in activity if one tyrosine is mutated and essential loss of all activity upon double mutation of the two tyrosines in question. It was found that a hydrogen bond from Tyr465 to the substrate oxygen is essential for controlling the regioselectivity of the reaction. Furthermore, a relationship between the presence of this hydrogen bond and the separation of reactants was found. Two groups of amino acid segments were identified each as moving collectively. Furthermore, an overall anti-correlation was found between the movements of these two individually collectively moving groups, made up by parts of the cap-region, including the two tyrosines, and the site of the catalytic triad, respectively. This overall anti-correlated collective domain motion is, perhaps, involved in the conversion of E.NAC to E.TS.     

Catalytic mechanism of limonene epoxide hydrolase, a theoretical study

The catalytic mechanism of limonene epoxide hydrolase (LEH) was investigated theoretically using the density functional theory method B3LYP. LEH is part of a novel limonene degradation pathway found in Rhodococcus erythropolis DCL14, where it catalyzes the hydrolysis of limonene-1,2-epoxide to give limonene-1,2-diol. The recent crystal structure of LEH was used to build a model of the LEH active site composed of five amino acids and a crystallographically observed water molecule. With this model, hydrolysis of different substrates was investigated. It is concluded that LEH employs a concerted general acid/general base-catalyzed reaction mechanism involving protonation of the substrate by Asp101, nucleophilic attack by water on the epoxide, and abstraction of a proton from water by Asp132. Furthermore, we provide an explanation for the experimentally observed regioselective hydrolysis of the four stereoisomers of limonene-1,2-epoxide.     

Possible involvement of collective domain movement in the catalytic reaction of soluble epoxide hydrolase

The hydrolysis of 1S,2S‐trans‐methylstyrene oxide by soluble epoxide hydrolases is studied by a 4‐ns molecular dynamics (MD) simulation. An analysis of the extent of correlated motions in the active site was carried out. Based on the calculated cross correlation coefficients form the covariance matrix, a new correlation parameter, termed the supercorrelation coefficient, is introduced. The supercorrelation coefficient indicates the extent to which two amino acid residues move in a correlated manner with respect to all other residues in the protein. The resulting map of the supercorrelation coefficients was used to identify segments of the protein that may show collective domain movements. Interestingly, an anti‐correlated motion is located across the active site, involving the catalytic triad and the tyrosines. This may suggest that if a link exists between enzyme dynamics and catalysis, it may be through an anti‐correlated collective domain movement that compresses the active site, thus initiating the conversion of E–NAC to E–TS. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004     

Insights into the cycloaddition reaction mechanism between ketenimine and unsaturated hydrocarbon: A theoretical study

The cycloaddition reaction mechanisms between interstellar molecule ketenimine and unsaturated hydrocarbon (ethyne and ethylene) have been systematically investigated employing the second-order Møller-Plesset perturbation theory (MP2) method. Geometry optimizations and vibrational analyses have been performed for the stationary points on the potential energy surfaces of the system. The calculated results show that it can be produced the five-membered cyclic carbene intermediates through pericyclic reaction processes between ketenimine and ethyne (or ethylene). For the reaction between ketenimine and ethyne, through the following H-transferred processes, carbene intermediate can be isomerized to the pyrrole compounds. For the reaction between ketenimine and ethylene, carbene intermediate can be isomerized to the pyrroline compounds. The present study is helpful to understand the reactivity of nitrogenous cumulene ketenimine and the formation of prebiotic species in interstellar space.     

Regio- and enantioselective hydrolysis of phenyloxiranes catalyzed by soluble epoxide hydrolase

The regio- and enantioselective hydrolysis of several phenyloxiranes catalyzed by soluble epoxide hydrolase (sEH) was investigated using recombinant human, mouse or cress sEH. Results indicate that human and mouse sEH enantioselectively hydrolyze (S,S)-alkyl-phenyloxiranes faster than the (R,R)-alkyl-phenyloxiranes investigated in this study, while cress sEH displayed opposite enantioselectivity. Preparation of pure (2R,3R)-3-phenylglycidol from the racemic mixture was achieved with a 31% yield using human sEH as catalyst. The sEH enzymes were found to be regioselective at the benzylic carbon of the phenyloxiranes, supporting the proposed mechanism in which one or more tyrosine residues in the active site of the enzyme act as a general acid catalyst in the alkylation half reaction.     

A theoretical insight into the reaction mechanism of photochemical transposition from pyrazole to imidazole

The mechanisms of the photochemical isomerization reactions were investigated by using a model system of 1,3,5-trimethylpyrazole ( 1) with the CASSCF (eight-electron/six-orbital active space) and MP2-CAS methods and the 6-311G(d) basis set. Three reaction pathways were examined in the present work. They are denoted as the ring-contraction-ring-expansion path (path I), the internal-cyclization-isomerization path (path II), and the conical-intersection path (path III). Our model investigations suggest that the preferred reaction route for the pyrazoles is as follows: reactant --> Franck-Condon region --> conical intersection --> photoproduct. In particular, the conical-intersection mechanism (path III) found in this work gives a better explanation than the previously proposed two other mechanisms (paths I and II). The theoretical findings also indicate that path III-1 should be favored over path III-2 from a kinetic point of view. This suggests that the quantum yield of 1,2,4-trimethylimidazole ( 2) should be greater than that of 1,2,5-trimethylimidazole ( 3), which supports the available experimental observations. Additionally, we propose a simple p-pi orbital topology model, which can be used as a diagnostic tool to predict the location of the conical intersections, as well as the geometries of the phototransposition products of various heterocycles.     

Insights into the mechanism and catalysis of the native chemical ligation reaction

Native chemical ligation of unprotected peptide segments involves reaction between a peptide-alpha-thioester and a cysteine-peptide, to yield a product with a native amide bond at the ligation site. Peptide-alpha-thioalkyl esters are commonly used because of their ease of preparation. These thioalkyl esters are rather unreactive so the ligation reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts used to date have been either a mixture of thiophenol/benzyl mercaptan, or the alkanethiol MESNA. Despite the use of these thiol catalysts, ligation reactions typically take 24-48 h. To gain insight into the mechanism of native chemical ligaton and in order to find a better catalyst, we investigated the use of a number of thiol compounds. Substituted thiophenols with pK(a) > 6 were found to best combine the ability to exchange rapidly and completely with thioalkyl esters, and to then act as effective leaving groups in reaction of the peptide-thioester with the thiol side chain of a cysteine-peptide. A highly effective and practical catalyst was (4-carboxylmethyl)thiophenol ('MPAA'), a nonmalodorous, water-soluble thiol. Use of MPAA gave an order of magnitude faster reaction in model studies of native chemical ligation and in the synthesis of a small protein, turkey ovomucoid third domain (OMTKY3). MPAA should find broad use in native chemical ligation and in the total synthesis of proteins.     

Insights into the mechanism of BN generation via boron triazide precursor: theoretical study

Potential-energy surfaces for unimolecular decomposition of B(N3)(3) have been studied to understand the possible mechanism for BN generation. The decomposition of B(N3)(3) takes place on either the singlet and triplet surface, and both processes are high exothermic and obey sequential mechanisms. For the singlet reaction, the rate-determining step corresponds to cleavage of the first azide bond and linear (1)NBNN instead of (1)BN was suggested as the dominant product at room temperature. For the triplet surface, a fragment process from (3)BN(7) to (3)BN(5) is predicted to be the rate-determining step; once this barrier is counteracted, the subsequent decomposition processes could easily occur to form final product (3)BN. In addition, the possible mechanism for generating BN film via B(N3)(3) was discussed based on MC-SCF calculation results. These findings might be helpful in understanding the controllable decomposition of B(N3)(3) as well as its application in generating BN films.     

A molecular model for the active site of S-adenosyl-l-homocysteine hydrolase

Summary S-adenosyl-l-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1.), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD⁺.Based on the 2.0 Å-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction.We also studied a model of the complex of AdoHcy hydrolase with NAD⁺, as well as the ternary complexes of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.     

Prospects for the inhibition of the phosphatase domain of human soluble epoxide hydrolase (sEH-P)

Russian Chemical Bulletin - This brief review highlights the history of the discovery of the phosphatase domain of human soluble epoxide hydrolase (sEH-P). The data on its structure and...     

A non-radical mechanism for methane hydroxylation at the diiron active site of soluble methane monooxygenase

We propose a non‐radical mechanism for the conversion of methane into methanol by soluble methane monooxygenase (sMMO), the active site of which involves a diiron active center. We assume the active site of the MMOH_Q intermediate, exhibiting direct reactivity with the methane substrate, to be a bis(μ‐oxo)diiron(IV ) complex in which one of the iron atoms is coordinatively unsaturated (five‐coordinate). Is it reasonable for such a diiron complex to be formed in the catalytic reaction of sMMO? The answer to this important question is positive from the viewpoint of energetics in density functional theory (DFT) calculations. Our model thus has a vacant coordination site for substrate methane. If MMOH_Q involves a coordinatively unsaturated iron atom at the active center, methane is effectively converted into methanol in the broken‐symmetry singlet state by a non‐radical mechanism; in the first step a methane C? H bond is dissociated via a four‐centered transition state (TS1) resulting in an important intermediate involving a hydroxo ligand and a methyl ligand, and in the second step the binding of the methyl ligand and the hydroxo ligand through a three‐centered transition state (TS2) results in the formation of a methanol complex. This mechanism is essentially identical to that of the methane–methanol conversion by the bare FeO⁺ complex and relevant transition metal–oxo complexes in the gas phase. Neither radical species nor ionic species are involved in this mechanism. We look in detail at kinetic isotope effects (KIEs) for H atom abstraction from methane on the basis of transition state theory with Wigner tunneling corrections.     

Insights into the reaction mechanism between propadienylidene and ethylene: a DFT study

The reaction mechanism between propadienylidene and ethylene has been systematically investigated employing the B3LYP/6-311++G** and MP2/cc-pVTZ levels of theory to better understand the reactivity of propadienylidene with unsaturated hydrocarbons. Geometry optimization, vibrational analysis, and energy property for the involved stationary points on the potential energy surface have been calculated. Two important initial reaction complexes characterized by three- and four-membered ring structures have been located firstly. After that, three different products possessing three-, four-, and five-membered ring characters have been obtained through three reaction pathways. In the first reaction pathway, a three-membered ring alkyne compound has been obtained. As for the second reaction pathway, it is a diffusion-controlled reaction, resulting in the formation of the four-membered ring conjugated diene compound. A five-membered conjugated diene compound has been obtained in the third reaction pathway, which is the most stable product in the available products thermodynamically. On the other hand, the second reaction pathway is the most favorable reaction to proceed kinetically.     

Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in reasonable yield. The enantioselectivity (E-value) of the resolution was calculated from progress curves for styrene oxide (E=16.2) and para-chlorostyrene oxide (E=32.2).     

Synthesis of a variety of optically active hydroxylated heterocyclic compounds using epoxide hydrolase technology

Novel epoxide hydrolases in Yarrowia lipolytica have been shown to hydrolyse a variety of functionalised epoxides with good to excellent stereoselectivity and at high volumetric productivities. Individual biotransformation products have been converted into optically active (R)-(tetrahydrofuran-2-yl)methanol (6), (S)-N-benzyl-3-hydroxypyrrolidine (7), (S)-3-hydroxytetrahydrothiophene (8), (S)-N-benzyl-3-acetoxypiperidine (10), (S)-3-hydroxytetrahydrofuran (16) and (R)-[(S)-N-benzylpyrrolidin-2-yl](phenyl)methanol (20).     

High-energy intermediate or stable transition state analogue: theoretical perspective of the active site and mechanism of beta-phosphoglucomutase

A recent crystal structure of beta-phosphoglucomutase from Lactococcus lactis is reported to contain a five-coordinate phosphorus with five oxygen ligands that is a high-energy reaction intermediate during the phosphoryl transfer in the isomerization of beta-glucose 1-phosphate to beta-glucose 6-phosphate. Subsequently, it has been suggested that this structure is a transition state analogue with a five-coordinate magnesium with two oxygen and three fluorine ligands. Two layer ONIOM(B3LYP:PM3MM) calculations have been performed to address the nature of this intermediate and the mechanism of the phosphoryl transfer. These calculations provide evidence that (1) the observed crystal structure is consistent with a five-coordinate magnesium (a stable transition state analogue), not a five-coordinate phosphorus (a phosphorane) as a high-energy intermediate, (2) the active site is stabilized by the extensive hydrogen-bonding network, (3) the transfer of the phosphoryl group proceeds through a moderate barrier (14 kcal mol-1) five-coordinate phosphorus without a stable phosphorane or metaphosphate intermediate, (4) this concerted transition state is directly coupled to a proton transfer from the oxygen of glucose to the carboxylic group of aspartate 10, and (5) a stable glucose 1,6-bis-phosphoglucose intermediate is formed.