[Effects of P and K fertilizer on content of coumarin and yield of Glehnia littoralis].

By a orthogonal experiment, the influence of different ratio of phosphorus and potassium fertilizers on imperatorin, isoimperatorin and psoralen contents and yield of Glehnia littoralis were studied. The results showed that root dry weight and the yield of G. littoralis increased when reasonably applied phosphorus fertilizer combined with potassium fertilizer within a certain range. And the influence of phosphorus fertilizer was greater than that of potassium fertilizer. The optimal value of root dry weight and yield achieved at both P2O5 360 kg x hm(-2), K2O 270 kg x hm(-2) and P2O5 360 kg x hm(-2), K2O 180 kg x hm(-2). The effects of different phosphorus and potassium treatments on the content of imperatorin, isoimperatorin and psoralen in G. littoralis were determined, which shows that the content increased with the moderate increase of phosphorus and potassium. And the effects of phosphorus fertilizer were more significantly. The isoimperatorin content achieved the largest value at P2O5 360 kg x hm(-2), K2O 270 kg x hm(-2), also a larger content of imperatorin and psoralen. The imperatorin content is the largest when applied P2O5 360 kg x hm(-2), K2O 180 kg x hm(-2), and the isoimperatorin content was higher as well. So that the treatment of P2O5 360 kg x hm(-2), K2O 180 kg x hm(-2) are suitable for promote to the agricultural production, which could improve the quality and yield of G. littoralis.

Franck-Condon factors perturbed by damped harmonic oscillators: solvent enhanced X (1)Ag ↔ A(1)B1u absorption and fluorescence spectra of perylene.

Damped harmonic oscillators are utilized to calculate Franck-Condon factors within displaced harmonic oscillator approximation. This is practically done by scaling unperturbed Hessian matrix that represents local modes of force constants for molecule in gaseous phase, and then by diagonalizing perturbed Hessian matrix it results in direct modification of Huang-Rhys factors which represent normal modes of solute molecule perturbed by solvent environment. Scaling parameters are empirically introduced for simulating absorption and fluorescence spectra of an isolated solute molecule in solution. The present method is especially useful for simulating vibronic spectra of polycyclic aromatic hydrocarbon molecules in which hydrogen atom vibrations in solution can be scaled equally, namely the same scaling factor being applied to all hydrogen atoms in polycyclic aromatic hydrocarbons. The present method is demonstrated in simulating solvent enhanced X (1)Ag ↔ A(1)B1u absorption and fluorescence spectra of perylene (medium-sized polycyclic aromatic hydrocarbon) in benzene solution. It is found that one of six active normal modes v10 is actually responsible to the solvent enhancement of spectra observed in experiment. Simulations from all functionals (TD) B3LYP, (TD) B3LYP35, (TD) B3LYP50, and (TD) B3LYP100 draw the same conclusion. Hence, the present method is able to adequately reproduce experimental absorption and fluorescence spectra in both gas and solution phases.

Theoretical studies on the reaction mechanism of PP1 and the effects of different oxidation states of the Mn-Mn center on the mechanism.

Protein phosphatase 1 (PP1) is a dinuclear metalloenzyme that catalyzes the dephosphorylation of serine and threonine residues. In this work, the catalytic reaction mechanism of PP1 was theoretically investigated by hybrid density functional theory. Firstly, an initial model of the Mn(II)-Mn(II) active site of PP1 was constructed on the basis of the high-resolution crystal structure, and stationary points along the reaction pathway were optimized and analyzed. The calculations provide strong support for the mechanism of the dephosphorylation by PP1 and suggest that His125 plays the role of donating a proton to the leaving group. Furthermore, reaction models with the Mn-Mn centers at different oxidation states [Mn(III)-Mn(II) and Mn(III)-Mn(III) centers] were designed. Our calculations show that increasing the oxidation state of one or both Mn(II) can shorten the bond lengths between the metal ions and the ligands, and increase the energy barrier of the related reactions. We found it interesting that artificially adding a negatively charged hydroxy ligand into the Mn(III)-Mn(II) center can recover the shortened coordination bonds and lower the increased energy barrier. Our investigation suggests that the definite oxidation states of the metal centers should be significantly correlated to the negative charges of the ligands not only in phosphoprotein phosphatases, but also in purple acid phosphatases and Escherichia coli 5'-nucleotidase. This means that all the members of phosphoprotein phosphatases adopt homodivalent centers, and suggests the heterovalent active sites of purple acid phosphatases have evolved from homodivalent ones.

Theoretical studies on the mechanism of activation of phosphoprotein phosphatases and purple acid phosphatases suggest an evolutionary strategy to survive in acidic environments.

Dephosphorylation reactions of phosphoprotein phosphatases (PPPs) share a common catalytic cycle. In one stage of the cycle, the active site is regenerated through formation of a new nucleophilic µ-hydroxy moiety and reprotonation of the proton donor, His125 (numbered according to the protein phosphatase 1 sequence). To date the exact details of the mechanism of this step remain uncertain. On the basis of recurring observations in several crystal structures, we propose an activation mechanism in which dephosphorylation of PPPs proceeds mainly through proton transfer from the water molecule that bridges the metal ions to His125, which is mediated by another water molecule. Our calculations using hybrid density functional theory and B3LYP functionals support this activation mechanism. We also propose that Asp95 facilitates proton transfer by eliminating the energy barrier and the backbone carbonyl oxygen atom of His248 acts mainly to orient and stabilize the µ-hydroxo (or water molecule) through hydrogen bonding. Furthermore, on the basis of the structural similarities of the active sites of purple acid phosphatases (PAPs) and PPPs, we speculate that PAPs are activated by a dual proton transfer mediated by one water molecule. Our calculations support this hypothesis and indicate that the active site of PAPs can still be active in an acidic environment (in agreement with the acid phosphatase activity of PAPs). Therefore, the variant of the activation mechanism from PPPs to PAPs implies an evolutionary adaptation to acidic environments.

Theoretical study of the oxidation of phenolates by the [Cu2O2(N,N'-di-tert-butylethylenediamine)2]2+ complex.

Experiments have shown that the µ-η(2):η(2)-peroxodicopper(II) complex [Cu(2)O(2)(N,N'-di-tert-butylethylenediamine)(2)](2+) rapidly oxidizes 2,4-di-tert-butylphenolate into a mixture of catechol and quinone and that, at the extreme temperature of -120 °C, a bis-µ-oxodicopper(III)-phenolate intermediate, labeled complex A, can be observed. These experimental results suggest a new mechanism of action for the dinuclear copper-containing enzyme tyrosinase, involving an early O-O bond-cleavage step. However, whether phenolate binding occurs before or after the cleavage of the O-O bond has not been possible to answer. In this study, hybrid density functional theory is used to study the synthetic reaction and, based on the calculated free-energy profile, a mechanism is suggested for the entire phenolate-oxidation reaction that agrees with the experimental observations. Most importantly, the calculations show that the very first step in the reaction is the cleavage of the O-O bond in the peroxo complex and that, subsequently, the phenolate substrate coordinates to one of the copper ions in the bis-µ-oxodicopper(III) complex to yield the experimentally characterized phenolate intermediate (A). The oxidation of the phenolate substrate into a quinone then occurs in three steps: 1) C-O bond formation, 2) coupled internal proton and electron transfer, and 3) electron transfer coupled to proton transfer from an external donor (acidic workup, experimentally). The first of these steps is rate limiting for the decay of complex A, with a calculated free-energy barrier of 10.7 kcal mol(-1) and a deuterium kinetic isotope effect of 0.90, which are in good agreement with the experimental values of 11.2 kcal mol(-1) and 0.83(±0.09). The tert-butyl substituents on both the phenol substrate and the copper ligands need to be included in the calculations to give a correct description of the reaction mechanism.

Mechanism of tungsten-dependent acetylene hydratase from quantum chemical calculations.

Acetylene hydratase is a tungsten-dependent enzyme that catalyzes the nonredox hydration of acetylene to acetaldehyde. Density functional theory calculations are used to elucidate the reaction mechanism of this enzyme with a large model of the active site devised on the basis of the native X-ray crystal structure. Based on the calculations, we propose a new mechanism in which the acetylene substrate first displaces the W-coordinated water molecule, and then undergoes a nucleophilic attack by the water molecule assisted by an ionized Asp13 residue at the active site. This is followed by proton transfer from Asp13 to the newly formed vinyl anion intermediate. In the subsequent isomerization, Asp13 shuttles a proton from the hydroxyl group of the vinyl alcohol to the α-carbon. Asp13 is thus a key player in the mechanism, but also W is directly involved in the reaction by binding and activating acetylene and providing electrostatic stabilization to the transition states and intermediates. Several other mechanisms are also considered but the energetic barriers are found to be very high, ruling out these possibilities.

Mechanistic insight into the N=N bond-cleavage of azo-compounds that was induced by an Al-Al-bonded compound [L(2-)Al(II)-Al(II)L(2-)].

An α-diimine-stabilized Al-Al-bonded compound [L(2-)Al(II)-Al(II)L(2-)] (L = [{(2,6-iPr(2)C(6)H(3))NC(Me)}(2)]; 1) consists of dianionic α-diimine ligands and sub-valent Al(2+) ions and thus could potentially behave as a multielectron reductant. The reactions of compound 1 with azo-compounds afforded phenylimido-bridged products [L(-)Al(III)(µ(2)-NPh)(µ(2)-NAr)Al(III)L(-)] (2-4). During the reaction, the dianionic ligands and Al(2+) ions were oxidized into monoanions and Al(3+), respectively, whilst the [NAr](2-) imides were produced by the four-electron reductive cleavage of the N=N double bond. Upon further reduction by Na, the monoanionic ligands in compound 2 were reduced to the dianion to give [(L(2-))(2)Al(III)(2)(µ(2)-NPh)(2)Na(2)(thf)(4)] (5). Interestingly, when asymmetric azo-compounds were used, the asymmetric adducts were isolated as the only products (compounds 3 and 4). DFT calculations indicated that the reaction was quite feasible in the singlet electronic state, but the final product with the triplet-state monoanionic ligands could result from an exothermic singlet-to-triplet conversion during the reaction process.

Comparison of the performance of exact-exchange-based density functional methods.

How to describe nondynamic electron correlation is still a major challenge to density functional theory (DFT). Recent models designed particularly for this problem, such as Becke'05 (B05) and Perdew-Staroverov-Tao-Scuseria (PSTS) functionals employ the exact-exchange density, the efficient calculation of which is technically quite challenging. We have recently implemented self-consistently the B05 functional based on an efficient resolution-identity (RI) technique. In this study, we report a self-consistent RI implementation of the PSTS functional. In contrast to its original implementation, our version brings no limitation on the choice of the basis set. We have also implemented the Mori-Sanchez-Cohen-Yang-2 (MCY2) functional, another recent DFT method that includes full exact exchange. The performance of PSTS, B05, and MCY2 is validated on thermochemistry, reaction barriers, and dissociation energy curves, with an emphasis on nondynamic correlation effects in the discussion. All three methods perform rather well in general, B05 and MCY2 being on average somewhat better than PSTS. We include also results with other functionals that represent various aspects of the development in this field in recent years, including B3LYP, M06-HF, M06-2X, ωB97X, and TPSSh. The performance of the heavy-parameterized functionals M06-2X and ωB97X is on average better than that of B05, MCY2, and PSTS for standard thermodynamic properties and reactions, while the latter functionals do better in hydrogen abstraction reactions and dissociation processes. In particular, B05 is found to be the only functional that yields qualitatively correct dissociation curves for two-center symmetric radicals like He(2)(+). Finally, we compare the performance of all these functionals on a strongly correlated exemplary case system, the NO dimer. Only PSTS, B05, and MCY2 describe the system qualitatively correctly. Overall, this new type of functionals show good promise of overcoming some of the difficulties DFT encounters for systems with strong nondynamic correlation.

Mechanism of mycolic acid cyclopropane synthase: a theoretical study.

The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.

Theoretical investigation of the first-shell mechanism of acetylene hydration catalyzed by a biomimetic tungsten complex.

The reaction mechanism of the hydration of acetylene to acetaldehyde catalyzed by [W(IV)O(mnt)(2)](2-) (where mnt(2-) is 1,2-dicyanoethylenedithiolate) is studied using density functional theory. Both the uncatalyzed and the catalyzed reaction are considered to find out the origin of the catalysis. Three different models are investigated, in which an aquo, a hydroxo, or an oxo coordinates to the tungsten center. A first-shell mechanism is suggested, similarly to recent calculations on tungsten-dependent acetylene hydratase. The acetylene substrate first coordinates to the tungsten center in an η(2) fashion. Then, the tungsten-bound hydroxide activates a water molecule to perform a nucleophilic attack on the acetylene, resulting in the formation of a vinyl anion and a tungsten-bound water molecule. This is followed by proton transfer from the tungsten-bound water molecule to the newly formed vinyl anion intermediate. Tungsten is directly involved in the reaction by binding and activating acetylene and providing electrostatic stabilization to the transition states and intermediates. Three other mechanisms are also considered, but the associated energetic barriers were found to be very high, ruling out those possibilities.

Three-step calcination synthesis of high-purity Li8ZrO6 with CO2 absorption properties.

Li(8)ZrO(6) contains a high lithium content and may bear a great ability of CO(2) absorption, yet the reports about the properties of CO(2) absorption on Li(8)ZrO(6) are few to date for its difficulty in production. In this paper, high-purity Li(8)ZrO(6) is synthesized via a three-step calcination method combined with an effective lithium source and a suitable initial Li/Zr molar ratio. The produced Li(8)ZrO(6) possesses a great CO(2) absorption capacity of about 53.98 wt % at 998 K, which could be well-maintained in a wide range of CO(2) partial pressures of 0.1-1.0 bar although it decreased gradually during the multicycle process of CO(2) absorption-desorption in a 10% CO(2) feed stream because of the high working temperature. These properties imply that Li(8)ZrO(6) may be a new option for high-temperature CO(2) capture applied in industrial processes such as a steam methane reformer.

DFT study of the asymmetric nitroaldol (Henry) reaction catalyzed by a dinuclear Zn complex.

We report the mechanism of asymmetric nitroaldol (Henry) reaction catalyzed by a dinuclear Zn complex using density functional theory. The experimentally proposed catalytic cycle is validated, in which the first step is the deprotonation of nitromethane by the ethyl anion of the catalyst, subsequently a C-C bond formation step, and then the protonation of the resulting alkoxide. Three mechanistic scenarios (differing in binding modes) have been considered for the C-C bond formation step. The origin of the enantioselectivity is discussed. Our calculations supported that the S configurations are the major products, which is in agreement with the experimental observations.

Differential diagnosis of pancreatic cancer from normal tissue with digital imaging processing and pattern recognition based on a support vector machine of EUS images.

BACKGROUND: EUS can detect morphologic abnormalities of pancreatic cancer with high sensitivity but with limited specificity. OBJECTIVE: To develop a classification model for differential diagnosis of pancreatic cancer by using a digital imaging processing (DIP) technique to analyze EUS images of the pancreas. DESIGN: A retrospective, controlled, single-center design was used. SETTING: The study took place at the Second Military Medical University, Shanghai, China. PATIENTS: There were 153 pancreatic cancer and 63 noncancer patients in this study. INTERVENTION: All patients underwent EUS-guided FNA and pathologic analysis. MAIN OUTCOME MEASUREMENTS: EUS images were obtained and correlated with cytologic findings after FNA. Texture features were extracted from the region of interest, and multifractal dimension vectors were introduced in the feature selection to the frame of the M-band wavelet transform. The sequential forward selection process was used for a better combination of features. By using the area under the receiver operating characteristic curve and other texture features based on separability criteria, a predictive model was built, trained, and validated according to the support vector machine theory. RESULTS: From 67 frequently used texture features, 20 better features were selected, resulting in a classification accuracy of 99.07% after being added to 9 other features. A predictive model was then built and trained. After 50 random tests, the average accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of pancreatic cancer were 97.98 ± 1.23%, 94.32 ± 0.03%, 99.45 ± 0.01%, 98.65 ± 0.02%, and 97.77 ± 0.01%, respectively. LIMITATIONS: The limitations of this study include the small sample size and that the support vector machine was not performed in real time. CONCLUSION: The classification of EUS images for differentiating pancreatic cancer from normal tissue by DIP is quite useful. Further refinements of such a model could increase the accuracy of EUS diagnosis of tumors.

Phosphate mono- and diesterase activities of the trinuclear zinc enzyme nuclease P1--insights from quantum chemical calculations.

Nuclease P1 is a trinuclear zinc enzyme that catalyzes the hydrolysis of single-stranded DNA and RNA. Density functional calculations are used to elucidate the reaction mechanism of this enzyme with a model of the active site designed on the basis of the X-ray crystal structure. 2-Tetrahydrofuranyl phosphate and methyl 2-tetrahydrofuranyl phosphate substrates are used to explore the phosphomonoesterase and phosphodiesterase activities of this enzyme, respectively. The calculations reveal that for both activities, a bridging hydroxide performs an in-line attack on the phosphorus center, resulting in inversion of the configuration. Simultaneously, the P-O bond is cleaved, and Zn2 stabilizes the negative charge of the leaving alkoxide anion and assists its departure. All three zinc ions, together with Arg48, provide electrostatic stabilization to the penta-coordinated transition state, thereby lowering the reaction barrier.

A density functional study of the structural and electronic properties of silicon monoxide clusters.

By use of density functional theory, a systemic theoretical study was conducted on the structural and electronic properties of ground-state silicon monoxide clusters ((SiO)(n), where n = 1-26). In our calculations, the most energetically favorable geometry for each cluster size was found to undergo a structural change from one dimension (linear) to three dimensions at cluster size n = 4, with the buckled structure as the favorable one. The sp(3) silicon containing structures are favorable for n = 5-13, and the Si-cored structures are energetically favorable at n = 14 and larger. Furthermore, for the lowest-energy structures obtained, the energy gaps between the highest occupied and lowest unoccupied molecular orbital, binding energies, ionization potentials, and electron affinities were calculated and analyzed to understand the evolutions in geometries and to identify any particularly stable species.

Why iron? A spin-polarized conceptual density functional theory study on metal-binding specificity of porphyrin.

Heme is a key cofactor of hemoproteins in which porphyrin is often found to be preferentially metalated by the iron cation. In our previous work [Feng, X. T.; Yu, J. G.; Lei, M.; Fang, W. H.; Liu, S. B. J. Phys. Chem. B 2009, 113, 13381], conceptual density functional theory (CDFT) descriptors have been applied to understand the metal-binding specificity of porphyrin. We found that the iron-porphyrin complex significantly differs in many aspects from porphyrin complexes with other metal cations except Ru, for which similar behaviors for the reactivity descriptors were discovered. In this study, we employ the spin-polarized version of CDFT to investigate the reactivity for a series of (pyridine)(n)-M(ll)-porphyrin complexes-where M = Mg, Ca, Cr, Mn, Co, Ni, Cu, Zn, Ru, and Cd, and n = 0, 1, and 2-to further appreciate the metal-binding specificity of porphyrin. Both global and local descriptors were examined within this framework. We found that, within the spin resolution, not only chemical reactivity descriptors from CDFT of the iron complex are markedly different from that of other metal complexes, but we also discovered substantial differences in reactivity descriptors between Fe and Ru complexes. These results confirm that spin properties play a highly important role in physiological functions of hemoproteins. Quantitative reactivity relationships have been revealed between global and local spin-polarized reactivity descriptors. These results contribute to our better understanding of the metal binding specificity and reactivity for heme-containing enzymes and other metalloproteins alike.

[3D visualization for simulating percutaneous transcatheter closure of atrial septal defect:a pilot clinical study].

OBJECTIVE: To evaluate the clinical value of 3D visualization method for simulating percutaneous transcatheter closure of atrial septal defect (ASD). METHODS: 3D volume render method was used for visualizing ASD and surrounding structures and 3D modeling method was applied for simulate the shape of occlusion device. The size and the distance between the lower edge of device and atria-ventricular valve of simulation occluder and actual selected atrial septal occluder (ASO) were compared in 30 patients underwent successful transcatheter closure of ASD. RESULTS: The location, geometry, size, extent of ASDs in children were displayed in 3D visualization. No significant difference was found between simulation occluder and ASO size measured from left atrium [(26.07 +/- 5.32) cm vs. (25.91 +/- 5.32) cm] and right atrium [(22.13 +/- 5.31) cm vs. (22.08 +/- 5.26) cm, all P > 0.05]. The distances from simulation occluder to mitral valves [(5.76 +/- 2.39) cm] and to tricuspid valves [(8.25 +/- 2.40) cm] were similar as ASO to atria-ventricular valves [(5.61 +/- 2.26) cm and (8.02 +/- 2.48) cm, respectively, all P > 0.05]. CONCLUSIONS: The simulating percutaneous transcatheter closure of ASD by 3D visualization technique could be a helpful noninvasive approach for ASO selection before the procedure of transcatheter occlusion of ASD.

Analysing and optimizing the electrolysis efficiency of a lithium cell based on the electrochemical and multiphase model.

Based on an electrochemical multiphysical simulation, a method for analysing electrolysis efficiency has been presented that considers the energy consumption required to produce a single kilogram of lithium and for the production of lithium, rather than the voltage in various parts. By adopting them as the criteria for analysing electrolysis efficiency in the lithium cell, several structural parameters have been optimized, such as the anode radius and anode-cathode distance. These parameters strongly affect the cell voltage and the velocity field distribution, which has a significant impact on the concentration distribution. By integrating the concentration distribution, the lithium production and energy consumption per kilogram, lithium is computed. By appointing the minimum of the chlorine and lithium concentration as the secondary reaction intensity, it is clear where the secondary reaction intensity is strong in the cell. The structure of a lithium electrolysis cell has been optimized by applying an orthogonal design approach, with the energy consumption notably decreasing from 35.0 to 28.3 kWh (kg Li)-1 and the lithium production successfully increasing by 0.17 mol.

Comprehensive analysis of the effect of rs2295080 and rs2536 polymorphisms within the mTOR gene on cancer risk.

There is still no conclusion on the potential effect of the rs2295080 and rs2536 polymorphisms of mTOR (mammalian target of rapamycin) gene on different cancers. Herein, we performed a comprehensive assessment using pooled analysis, FPRP (false-positive report probability), TSA (trial sequential analysis), and eQTL (expression quantitative trait loci) analysis. Eighteen high-quality articles from China were enrolled. The pooled analysis of rs2295080 with 9502 cases and 10,965 controls showed a decreased risk of urinary system tumors and specific prostate cancers [TG vs. TT, TG+GG vs. TT and G vs. T; P<0.05, OR (odds ratio) <1]. FPRP and TSA data further confirmed these results. There was an increased risk of leukemia [G vs. T, GG vs. TT, and GG vs. TT+TG genotypes; P<0.05, OR>1]. The eQTL data showed a potential correlation between the rs2295080 and mTOR expression in whole blood samples. Nevertheless, FPRP and TSA data suggested that more evidence is required to confirm the potential role of rs2295080 in leukemia risk. The pooled analysis of rs2536 (6653 cases and 7025 controls) showed a significant association in the subgroup of "population-based" control source via the allele, heterozygote, dominant, and carrier comparisons (P<0.05, OR>1). In conclusion, the TG genotype of mTOR rs2295080 may be linked to reduced susceptibility to urinary system tumors or specific prostate cancers in Chinese patients. The currently data do not strongly support a role of rs2295080 in leukemia susceptibility. Large sample sizes are needed to confirm the potential role of rs2536 in more types of cancer.

DFT study on the mechanism of Escherichia coli inorganic pyrophosphatase.

Escherichia coli inorganic pyrophosphatase (E-PPase) is a tetranuclear divalent metal dependent enzyme that catalyzes the reversible interconversion of pyrophosphate (PPi) and orthophosphate (Pi), with Mg(2+) conferring the highest activity. In the present work, the reaction mechanism of E-PPase is investigated using the hybrid density functional theory (DFT) method B3LYP with a large model of the active site. Our calculated results shed further light on the detailed reaction mechanism. In particular, the important residue Asp67, either protonated or unprotonated, was taken into account in the present calculations. Our calculations indicated that a protonated Asp67 is crucial for the reverse reaction to take place; however, it is lost sight of in the forward reaction. The bridging hydroxide is shown to be capable of performing nucleophilic in-line attack on the substrate from its bridging position in the presence of four Mg(2+) ions. During the catalysis, the roles of the four magnesium ions are suggested to provide a necessary conformation of the active site, facilitate the nucleophile formation and substrate orientation, and stabilize the trigonal bipyramid transition state, thereby lowering the barrier for the nucleophilic attack.