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     

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.     

Computational study of phosphatase activity in soluble epoxide hydrolase: high efficiency through a water bridge mediated proton shuttle

Recently, a new branch of fatty acid metabolism has been opened by the novel phosphatase activity found in the N-terminal domain of the, hence bifunctional, soluble epoxide hydrolase (sEH). Importantly, this finding has also provided a new site for drug targeting in sEH's activity regulation. Classical MD and hybrid Car-Parrinello QM/MM calculations have been performed to investigate the reaction mechanism of the phosphoenzyme intermediate formation in the first step of the catalysis. The results support a concerted multi-event reaction mechanism: (1) a dissociative in-line nucleophilic substitution for the phosphoryl transfer reaction; (2) a double proton transfer involved in the formation of a good leaving group in the transition state. The presence of a water bridge in the substrate/enzyme complex allowed an efficient proton shuttle, showing its key role in speeding up the catalysis. The calculated free energy of the favored catalytic pathway is approximately 19 kcal/mol, in excellent agreement with experimental data.     

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.     

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.     

Synthesis of adamantyl-containing 1,3-disubstituted diureas and thioureas,efficient targeted inhibitors of human soluble epoxide hydrolase

A series of adamantyl-containing 1,3-disubstituted diureas and thioureas containing different spacers between the ureylene group and the adamantyl substituent has been synthesized, their inhibitory activity against mammalian and human soluble epoxide hydrolase (sEH, E.C. 3.3.2.10) has been examined. The compounds synthesized were found to exhibit high inhibitory activity on the 0.4–2.8 nmol L^?1 level. The dependence between the inhibitor structure and its activity was established     

Insights into the reaction mechanism of soluble epoxide hydrolase from theoretical active site mutants

Density functional theory calculations of active site mutants are used to gain insights into the reaction mechanism of the soluble epoxide hydrolases (sEHs). The quantum chemical model is based on the X-ray crystal structure of the human soluble epoxide hydrolase. The role of two conserved active site tyrosines is explored through in silico single and double mutations to phenylalanine. Full potential energy curves for hydrolysis of (1S,2S)-beta-methylstyrene oxide are presented. The results indicate that the two active site tyrosines act in concert to lower the activation barrier for the alkylation step. For the wild-type and three different tyrosine mutant models, the regioselectivity of epoxide opening is compared for the substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide. An additional part of our study focuses on the importance of the catalytic histidine for the alkylation half-reaction. Different models are presented to explore the protonation state of the catalytic histidine in the alkylation step and to evaluate the possibility of an interaction between the nucleophilic aspartate and the catalytic histidine.     

Enhancing the enantioselectivity of an epoxide hydrolase by directed evolution

[reaction: see text] The epoxide hydrolase (EH) from Aspergillus niger, which shows a selectivity factor of only E = 4.6 in the hydrolytic kinetic resolution of glycidyl phenyl ether, has been subjected to directed evolution for the purpose of enhancing enantioselectivity. After only one round of error-prone polymerase chain reaction (epPCR), enantioselectivity was more than doubled (E = 10.8). The improved mutant enzyme contains three amino acid exchanges, two of which are spatially far from the catalytically active center.     

One-step preparation method for adamantyl-containing isocyanates,precursors of epoxide hydrolase inhibitors

One-step preparation method was developed for adamantyl-containing isocyanates obtained by Curtius reaction from adamantanecarboxylic and acetic acids. Synthesis of isocyanate was performed without the isolation of intermediate acid chloride which solution was added to sodium azide in boiling toluene. In the reactor the acid azide formation and its rearrangement in isocyanate occurred simultaneously. Yields of target products were 83–94% with respect to the acid.     

High-performance thin-layer chromatography assay for epoxide hydrolase activity and the determination of phenoxypropane-1,2-diols

A simple, rapid and sensitive high-performance thin-layer chromatographic assay for the determination of epoxide hydrolase activity in rat liver homogenates is described. It is extended to the determination of a series of phenoxypropane-1,2-diols. The hydrolase assay has the advantages of using a readily available substrate, 2,3-epoxypropyl 4-methoxyphenyl ether, of multiple sample application, and of the simultaneous determination of reaction product (diol) as well as unchanged substrate (epoxide). The use of an internal standard, 4-nitroanisole, results in high sensitivity and good reproducibility of the proposed method. The limit of diol detection is 20 pmol.     

Preparation of a very stable immobilized Solanum tuberosum epoxide hydrolase

The covalent immobilization of the Solanum tuberosum epoxide hydrolase (StEH) was explored using highly activated Sepabeads-epoxy or Glyoxyl-agarose based supports. A Glyoxyl-agarose immobilizate, prepared under optimized experimental conditions, led to a material exhibiting excellent thermal and chemical stability. The key step of this immobilization process was the use of 164 kDa dextran as an additive during immobilization, which prevented the enzyme from inactivation at the high pH (pH 10) necessarily used for performing this immobilization. This afforded a Glyoxyl-agarose-StEH immobilizate with 80% initial enzymatic activity retention and a stabilization factor of at least 300 at 60 °C, as compared to the free enzyme. The high enantio- and regio-selectivity properties of this novel biocatalyst were shown to be nearly identical to those of the free enzyme.     

Synthesis and properties of 1-(R-adamant-1-yl)-3-(1-propionylpiperidin-4-yl)ureas and 4-({4-[3-(R-adamant-1-yl)ureido]cyclohexyl}oxy)benzoic acids,efficient target-oriented human soluble epoxide hydrolase inhibitors

A reaction of R-adamant-1-yl isocyanates with 4-[(4-aminocyclohexyl)oxy]benzoic acid and 1-(4-aminopiperidin-1-yl)propan-1-one in DMF afforded corresponding ureas in 90—95% yield, the target-oriented inhibitors of epoxide hydrolase sEH. The ureas are characterized by reduced melting points and increased solubility in water, as well as by inhibitory activity in the range of 0.5—4.0 nmol L^–1.     

The roles of cysteines in the heme domain of human soluble guanylate cyclase

Soluble guanylate cyclase(sGC) is a critical heme-containing enzyme involved in NO signaling.The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striking and an indistinct issue.The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein.The site-directed mutations of three conserved cysteines(C78A,C122A and C174S) were studied systematically and the three mutants were characterized by gel filtration analysis,UV-vis spectroscopy and heme transfer examination.Cys78 was involved in heme binding but not referred to the dimerization,while Cys174 was demonstrated to be involved in the homodimerization.These results provide new insights into the cysteine-related dimerization regulation of sGC.