Substrate polarization in enzyme catalysis: QM/MM analysis of the effect of oxaloacetate polarization on acetyl-CoA enolization in citrate synthase

Citrate synthase is an archetypal carbon-carbon bond forming enzyme. It promotes the conversion of oxaloacetate (OAA) to citrate by catalyzing the deprotonation (enolization) of acetyl-CoA, followed by nucleophilic attack of the enolate form of this substrate on OAA to form a citryl-CoA intermediate and subsequent hydrolysis. OAA is strongly bound to the active site and its alpha-carbonyl group is polarized. This polarization has been demonstrated spectroscopically, [(Kurz et al., Biochemistry 1985;24:452-457; Kurz and Drysdale, Biochemistry 1987;26:2623-2627)] and has been suggested to be an important catalytic strategy. Substrate polarization is believed to be important in many enzymes. The first step, formation of the acetyl-CoA enolate intermediate, is thought to be rate-limiting in the mesophilic (pig/chicken) enzyme. We have examined the effects of substrate polarization on this key step using quantum mechanical/molecular mechanical (QM/MM) methods. Free energy profiles have been calculated by AM1/CHARMM27 umbrella sampling molecular dynamics (MD) simulations, together with potential energy profiles. To study the influence of OAA polarization, profiles were calculated with different polarization of the OAA alpha-carbonyl group. The results indicate that OAA polarization influences catalysis only marginally but has a larger effect on intermediate stabilization. Different levels of treatment of OAA are compared (MM or QM), and its polarization in the protein and in water analyzed at the B3LYP/6-31+G(d)/CHARMM27 level. Analysis of stabilization by individual residues shows that the enzyme mainly stabilizes the enolate intermediate (not the transition state) through electrostatic (including hydrogen bond) interactions: these contribute much more than polarization of OAA.     

Second step of hydrolytic dehalogenation in haloalkane dehalogenase investigated by QM/MM methods

Mechanistic studies on the hydrolytic dehalogenation catalyzed by haloalkane dehalogenases are of importance for environmental and industrial applications. Here, Car-Parrinello (CP) and ONIOM hybrid quantum-mechanical/molecular mechanics (QM/MM) are used investigate the second reaction step of the catalytic cycle, which comprises a general base-catalyzed hydrolysis of an ester intermediate (EI) to alcohol and free enzyme. We focus on the enzyme LinB from Sphingomonas paucimobilis UT26, for which the X-ray structure at atomic resolution is available. In agreement with previous proposals, our calculations suggest that a histidine residue (His272), polarized by glutamate (Glu132), acts as a base, accepting a proton from the catalytic water molecule and transferring it to an alcoholate ion. The reaction proceeds through a metastable tetrahedral intermediate, which shows an easily reversed reaction to the EI. In the formation of the products, the protonated aspartic acid (Asp108) can easily adopt conformation of the relaxed state found in the free enzyme. The overall free energy barrier of the reaction calculated by potential of the mean force integration using CP-QM/MM calculations is equal to 19.5 +/- 2 kcal . mol(-1). The lowering of the energy barrier of catalyzed reaction with respect to the water reaction is caused by strong stabilization of the reaction intermediate and transition state and their preorganization by electrostatic field of the enzyme.     

Copper binding sites in the C-terminal domain of mouse prion protein: A hybrid (QM/MM) molecular dynamics study

We present a hybrid QM/MM Car-Parrinello molecular dynamics study of the copper-loaded C-terminal domain of the mouse prion protein. By means of a statistical analysis of copper coordination in known protein structures, we localized the protein regions with the highest propensity for copper ion binding. The identified candidate structures were subsequently refined via QM/MM simulations. Their EPR characteristics were computed to make contact with the experimental data and to probe the sensitivity to structural and chemical changes. Overall best agreement with the experimental EPR data (Van Doorslaer et al., J Phys Chem B 2001; 105: 1631-1639) and the information currently available in the literature is observed for a binding site involving H187. Moreover, a reinterpretation of the experimental proton hyperfine couplings was possible in the light of the present computational findings.     

A QM/MM study of the reaction mechanism of (R)‐hydroxynitrile lyases from Arabidopsis thaliana (AtHNL)

Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R‐selective HNL enzyme containing an α/β‐hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1‐C2 bond and the rotation of the generated cyanide ion (CN^?) follow, afterwards, CN^? abstracts a proton from His236 via Ser81. The C1‐C2 bond cleavage and the protonation of CN^? correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol^?1), suggesting that both of the two processes contribute a lot to rate‐limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1‐C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol^?1. The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN^? is stabilized by the hydroxyl group of Ser81 and the main‐chain NH‐groups of Ala13 and Phe82. Proteins 2015; 83:66–77. © 2014 Wiley Periodicals, Inc.     

MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method

MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase), a three-domain bacterial protein, catalyses a highly specific incorporation of D-glutamate to the cytoplasmic intermediate UDP-N-acetyl-muramoyl-L-alanine (UMA) utilizing ATP hydrolysis to ADP and P(i). This reaction is part of a biosynthetic path yielding bacterial peptidoglycan. On the basis of structural studies of MurD complexes, a stepwise catalytic mechanism was proposed that commences with a formation of the acyl-phosphate intermediate, followed by a nucleophilic attack of D-glutamate that, through the formation of a tetrahedral reaction intermediate and subsequent phosphate dissociation, affords the final product, UDP-N-acetyl-muramoyl-L-alanine-D-glutamate (UMAG). A hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling approach was utilized, combining the B3LYP QM level of theory with empirical force field simulations to evaluate three possible reaction pathways leading to tetrahedral intermediate formation. Geometries of the starting structures based on crystallographic experimental data and tetrahedral intermediates were carefully examined together with a role of crucial amino acids and water molecules. The replica path method was used to generate the reaction pathways between the starting structures and the corresponding tetrahedral reaction intermediates, offering direct comparisons with a sequential kinetic mechanism and the available structural data for this enzyme. The acquired knowledge represents new and valuable information to assist in the ongoing efforts leading toward novel inhibitors of MurD as potential antibacterial drugs.     

基于密度泛函理论研究Watson–Crick碱基对中的氢键特征(英文)

基于密度泛函理论(DFT)研究腺嘌呤、胸腺嘧啶、鸟嘌呤、胞嘧啶以及腺嘌呤胸腺嘧啶碱基对、鸟嘌呤胞嘧啶碱基对。在DFT-B3LYP/6-31G**水平上利用自然键轨道理论分析研究结果显示,互补碱基对的结构和电子特征有利于氢键的形成。本文中讨论几何结构、电子结构、分子轨道和能量对于氢键形成的影响。此研究结果将有助于更好的理解AT和GC碱基对中氢键与它们的结构特性之间的关系。     

Structure,stability and water permeation of ([D-Leu-L-Lys-(D-Gln-L-Ala)3]) cyclic peptide nanotube: a molecular dynamics study

The structural stability of 8 × ([D-Leu-L-Lys-(D-Gln-L-Ala)₃]) cyclic peptide nanotube (CPN) in water and different phospholipid bilayers were explored by 100 ns independent molecular dynamics (MD) simulations. The role of non-bonded interaction energy between the side and main chains of cyclic peptide rings in different membrane environments assessed, wherein the repulsive electrostatic interaction energy between neighbouring cyclic peptide rings was found adequate to break hydrogen bond energy thereby to crumple CPN. Further, the water permeation across the CPN channel was studied in four types of phospholipid bilayers- DMPG (1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), POPC (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) from MD simulations. DMPS membrane shows higher non-bonded interaction energies (?1913.06 kJ/mol of electrostatic interaction energy and ?994.13 kJ/mol of van der Waals interaction energy) with CPN due to the presence of polar molecules in lipid structure. Thusly, the non-bonded interaction energies were essential towards the stability of CPN than hydrogen bonds between the nearby cyclic peptides. The result also reveals the role of side chains, hydrogen bonds and non-bonded interaction energies in an aqueous environment. The diffusion coefficient of water obtained from means square deviation calculation shows similar coefficients irrespective of the lipid surroundings. However, the permeation coefficients demonstrate water flow in the channel relies upon the environment.     

Investigation of the inhibition pathway of glucosamine synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid by semiempirical quantum mechanical and molecular mechanics methods

Glucosamine synthase (E.C. 2.6.1.16) is a promising target in antifungal drug design. It has been reported that its potent inhibitor, N³-(4-methoxyfu-maroyl)-L-2,3-diaminopropanoic acid (FMDP), inactivates the enzyme by the Michael addition of the S-H group to the FMDP molecule followed by cyclisation reactions. In this study we have investigated, by means of semiempirical MNDO, PM3 and molecular mechanics methods, the energetics and kinetic possibility of the formation of various stereoisomers of the products of cyclisation of the Michael addition products detected experimentally. It was found that the substituted 1,4-thiazin-3-one can be formed in one step under alkaline conditions; the stereoisomers of this compound predicted to be the most stable on the basis of theoretical calculations are also the dominant ones in reality.Abbreviations FMDP N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid - MNDO Modified Neglect of Diatomic Overlap - AM1 Advanced Method 1 - PM3 (full abbreviation: MNDOPM3) Modified Neglect of Diatomic Overlap, Parametrised Method 3 Correspondence to: A. Liwo     

The nature of tryptophan radicals involved in the long-range electron transfer of lignin peroxidase and lignin peroxidase-like systems: Insights from quantum mechanical/molecular mechanics simulations

A catalytically active tryptophan radical has been demonstrated to be involved in the long-range electron transfer to the heme cofactor of lignin peroxidase (LiP) from Phanerochaete chrysosporium although no direct detection by EPR spectroscopy of the tryptophan radical intermediate has been reported to date. An engineering-based approach has been used to manipulate the microenvironment of the redox-active tryptophan site in LiP and Coprinus cinereus Peroxidase (CiP), allowing the direct evidence of the tryptophan radical species. In light of the newly available EPR experimental data, we performed a quantum mechanical/molecular mechanics computational study to characterize the tryptophan radicals in the above protein matrices as well as in pristine LiP. The nature of the tryptophan radicals is discussed together with the analysis of their environment with the aim of understanding the different behavior of pristine LiP in comparison with that of LiP and CiP variants.     

中国茜草属sect.Oligoneura的形态计量学研究

茜草属为茜草科模式属,全球约80种,中国有近一半的物种,是茜草属的重要分布中心,大部分隶属于sect.Oligoneura。但是,该组内各种间的形态特征错综复杂,依靠单个性状不易划定种间界限。该研究在全面衡量sect.Oligoneura组内各种间形态特征的基础上,选取该组20种共171份标本,对其3个数量性状和28个定性性状进行聚类分析和主成分分析。结果表明:在主成分分析中,前3个主成分累积贡献率有46.88%,性状的累积贡献率增长并不明显,但该结果仍与聚类分析结果一致。对前3个主成分贡献较大的特征包括叶形(叶长宽比)、叶柄长度、叶脉、每轮叶片数和生活型等,这些形态性状也是该属传统分类学中重要的物种鉴定特征。该研究结果与分子系统发育结果基本一致,支持该组内ser.Chinenses、R.mandersii group、R.angustissima group和R.siamensis group类群的划分,但并不支持ser.Cordifoliae单独成系。sect.Oligoneura内除少数物种外,大部分物种界限均比较清晰。形态计量学研究为sect.Oligoneura甚至茜草属的物种鉴定与划分提供了重要的参考意义。     

Comparison of a QM/MM force field and molecular mechanics force fields in simulations of alanine and glycine "dipeptides" (Ace-Ala-Nme and Ace-Gly-Nme) in water in relation to the problem of modeling the unfolded peptide backbone in solution

We compare the conformational distributions of Ace-Ala-Nme and Ace-Gly-Nme sampled in long simulations with several molecular mechanics (MM) force fields and with a fast combined quantum mechanics/molecular mechanics (QM/MM) force field, in which the solute's intramolecular energy and forces are calculated with the self-consistent charge density functional tight binding method (SCCDFTB), and the solvent is represented by either one of the well-known SPC and TIP3P models. All MM force fields give two main states for Ace-Ala-Nme, beta and alpha separated by free energy barriers, but the ratio in which these are sampled varies by a factor of 30, from a high in favor of beta of 6 to a low of 1/5. The frequency of transitions between states is particularly low with the amber and charmm force fields, for which the distributions are noticeably narrower, and the energy barriers between states higher. The lower of the two barriers lies between alpha and beta at values of psi near 0 for all MM simulations except for charmm22. The results of the QM/MM simulations vary less with the choice of MM force field; the ratio beta/alpha varies between 1.5 and 2.2, the easy pass lies at psi near 0, and transitions between states are more frequent than for amber and charmm, but less frequent than for cedar. For Ace-Gly-Nme, all force fields locate a diffuse stable region around phi = pi and psi = pi, whereas the amber force field gives two additional densely sampled states near phi = +/-100 degrees and psi = 0, which are also found with the QM/MM force field. For both solutes, the distribution from the QM/MM simulation shows greater similarity with the distribution in high-resolution protein structures than is the case for any of the MM simulations.     

Associations between blood metal/ metalloid concentration and human semen quality and sperm function: A cross-sectional study in Hong Kong

BackgroundThe association between metal/metalloid exposure on human sperm quality is still inconclusive. There is a lack of data on the effect of metal/metalloid on sperm function.MethodsThe aim of this study was to clarify the association between blood metal/metalloid concentration and traditional and functional sperm parameters, the blood concentration of Pb, Hg, Cd, As, Ni, Mo, Zn, Cu, Se, Fe, Mg, Cr and Ca of 288 men in Hong Kong were assessed by inductively coupled plasma-mass spectrometry, and sperm parameters including sperm concentration, motility, morphology, vitality, total sperm count, total motile sperm count, sperm DNA fragmentation and sperm acrosome reaction were measured. Demographic and lifestyle questionnaires were also provided for all participants. Multivariable linear regression analysis was performed to test the association between blood metal/ metalloid concentration and semen parameters after adjusting for relevant confounding variables.ResultsThe results showed that moderate to high level of blood Pb concentration (>27.19 μg/L) appeared to be negatively associated with sperm morphology (P < 0.05); high level of blood Cd concentration (>1.44 μg/L) was negatively associated with sperm acrosome reaction (P < 0.05); Mo was positively associated with semen volume (P < 0.05), however, high level of blood Mo concentration (>13.52 μg/L) was negatively associated with sperm vitality (P < 0.05); high level of blood Zn concentration (>6.20 mg/L) was positively associated with sperm vitality (P < 0.05); moderate level of blood Fe concentration (526.89−566.63 mg/L) was positively associated with sperm acrosome reaction (P < 0.05); moderate level of blood Ca concentration (55.92−66.10 mg/L) was positively associated with semen volume (P < 0.05); however, lower level of blood Ca concentration (45.90−55.92 mg/L) was negatively associated with sperm morphology (P < 0.05).ConclusionsOur results suggested that the sperm function could be affected by blood Cd and Fe concentration and traditional sperm parameters could be affected by blood concentration of Mo, Zn, Pb and Ca.     

A phenetic study of Cassia sensu lato (Leguminosae-Caesalpinioideae: Cassieae: Cassiinae) in Thailand

Cassia L. sensu lato, a large heterogeneous genus of flowering plants, occurs naturally in the tropics around the world. Recent works based on floral morphology have supported a division of this genus into three genera, namely Cassia L. s. str., Chamaecrista Moench and Senna Mill. In order to investigate this new classification, 508 specimens of 18 taxa of the genus Cassia s.l. grown in Thailand were analyzed using cluster analysis and canonical discriminant analysis. The total 32 vegetative and reproductive morphological characters were employed in these analyses. In cluster analysis, Cassia s.l. can be separated into four groups, respectively viz. Chamaecrista, Senna alata, Senna and Cassia s /str. The four-cluster grouping is discussed. From a canonical discriminant analysis using the four-cluster grouping as a priori groups, it can be concluded that Cassia s. str., Senna, and Chamaecrista are indeed distinct taxa. The three most important characters that separate the three genera are filament length, fruit length, and ovary stalk length. These quantitative characters, together with some qualitative characters, were useful in constructing an identification key to these genera. Among the three genera, it was also found that Senna is rather a heterogeneous taxon. The difference between the studied species was discussed.     

Clonal reproduction, vegetative multiplication and habitat colonisation in Tussilago farfara (Asteraceae): A combined morpho-ecological and molecular study

Using a combined morpho-ecological and molecular (amplified fragment length polymorphism (AFLP) fingerprinting) approach, clonal growth, clonal reproduction and the resulting vegetative multiplication were examined in the pioneer species Tussilago farfara (Asteraceae).

In two forest plots in Berlin (Germany), all modules and spacers of the plant were mapped and dug out. The plants showed intense clonal growth with leaf modules connected through long branched rhizomes. The rhizomes were brittle and regularly exhibited rotting zones and breaks. This clonal reproduction can occur rather fast; in a submersed stand in Lanke (Brandenburg, Germany), rhizomes showed signs of disintegration only a few weeks after the modules developed. As a consequence, modules become separated and genets fragment into merigenets.

Using AFLP fingerprints, the relationships of samples from the two dug out plots were assessed: 15 and 18 analysed samples (representing 13 and 16 merigenets) belonged to only two and three distinct genets, respectively. Each of these genets was split into at least two to eleven morphologically independent merigenets, indicating frequent and effective clonal reproduction.

The study highlights the relevance of vegetative multiplication in T. farfara, achieved rapidly through repeated clonal growth and subsequent clonal reproduction. The typical strategy of habitat colonisation and maintenance is described and illustrated for this pioneer species.     

Evolution in Carex L. sect. Spirostachyae (Cyperaceae): A molecular and cytogenetic approach

Carex sect. Spirostachyae comprises 25 species displaying the centre of diversity in Eurasia. Phylogenetic analysis of the ITS nrDNA region of 20 species of sect. Spirostachyae, six species of sect. Ceratocystis, five species of sect. Elatae, and eight outgroup species reveals that neither sect. Spirostachyae nor sect. Elatae is monophyletic. With the exclusion of Carex cretica, the 19 species of sect. Spirostachyae studied form a clade with the five species of tropical-subtropical sect. Elatae. Taxonomy of the core Spirostachyae is not only mostly in agreement with our phylogenetic hypothesis, but also with ecological and new cytogenetic results. Two main groups with different chromosome numbers and edaphic preferences are identified in the core Spirostachyae. One includes primarily acidophilous species with high chromosome numbers (2n=(64)68–84), whereas the other one includes mainly basophilous species with lower chromosome numbers (2n=60–74(75)). Chromosome-number variation is extremely different in the core Spirostachyae, showing great stability in some widespread species (e.g. Carex extensa) but an active chromosome evolution – faster chromosomal rearrangements, fusion and fission events than ITS nucleotide substitutions – in more restricted species (e.g. Carex troodi). Biogeography of the two amphiatlantic pairs of species reveals two independent colonizations of South America from the European continent. The geographical barrier of the Strait of Gibraltar has played different roles in the course of evolution of this section, acting as an effective barrier to gene flow in one case (Carex helodes) but as a limited barrier or recent separation in two others (Carex distans, Carex punctata).