Mechanistic Insights for Formation of an Organometallic Co–C Bond in the Methyl Transfer Reaction Catalyzed by Methionine Synthase

Methionine synthase (MetH) catalyzes the transfer of a methyl group from methyltetrahydrofolate (CH₃–H₄Folate) to the cob­(I)­alamin intermediate to form an organometallic Co–C bond, a reaction similar to that of CH₃–H₄Folate:corrinoid/iron–sulfur protein (CFeSP) methyltransferase (MeTr). How precisely it is formed remains elusive because the displacement of a methyl group from the tertiary amine is not a facile reaction. To understand the electronic structure and mechanistic details of the MetH–cob­(I)­alamin:CH₃–H₄Folate reaction complex, we applied quantum mechanics/molecular mechanics (QM/MM) computations. The hybrid QM/MM calculations reveal the traditionally assumed S_N2 mechanism for formation the CH₃–cob­(III)­alamin resting state where the activation energy barrier for the S_N2 reaction was found to be ∼8–9 kcal/mol, which is comparable with respect to the determined experimental rate constant. However, the possibility of an electron transfer (ET) based radical mechanism consistent with the close-lying diradical states observed from triplet and open-shell singlet states has also been suggested as an alternative, where first an electron transfer from His-on cob­(I)­alamin to the pterin ring of the protonated CH₃–H₄Folate takes place, forming the Co^II(d⁷)–pterin radical (π*)¹ diradical state, followed by a methyl radical transfer. Although the predicted energy barrier for the ET-mediated radical reaction is comparable to that of the S_N2 pathway, the major advantage of ET is that a methyl radical can be transferred at a longer distance, which does not require the close proximity of two binding modules of MetH as does the S_N2 type. In addition, based on the energy barrier of the transition state (TS) in both the protonated (∼8–9 kcal/mol) and the unprotonated N5 (39 kcal/mol) species of the CH₃–H₄Folate, it can be inferred that the protonation event must takes place either prior to or during the methyl transfer reaction in a ternary complex. The results of the present study including mechanistic insights can have implications to a broad class of corrinoid–methyltransferases, which utilize a CH₃–H₄Folate substrate or its related analogues as methyl donor

USES OF SAMPLING TECHNIQUES & INVENTORY CONTROL WITH CAPACITY CONSTRAINTS

The main aim of the present book is to suggest some improved estimators using auxiliary and attribute information in case of simple random sampling and stratified random sampling and some inventory models related to capacity constraints. <br

Metal-Free Transfer Hydrogenation of Nitroarenes in Water with Vasicine: Revelation of Organocatalytic Facet of an Abundant Alkaloid

Vasicine, an abundantly available quinazoline alkaloid from the leaves of <i>Adhatoda vasica</i>, has been successfully employed for metal- and base-free reduction of nitroarenes to the corresponding anilines in water. The method is chemoselective and tolerates a wide range of reducible functional groups, such as ketones, nitriles, esters, halogens, and heterocyclic rings. Dinitroarenes reduced selectively to the corresponding nitroanilines under the present reaction conditions

Charge Separation Propensity of the Coenzyme B₁₂–Tyrosine Complex in Adenosylcobalamin-Dependent Methylmalonyl–CoA Mutase Enzyme

We report the electrophilic Fukui function analysis based on density functional reactivity theory (DFRT) to demonstrate the feasibility of the proton-coupled electron transfer (PCET) mechanism. To characterize the charge propensity of an electron-transfer site other than the proton-acceptor site of the coenzyme B₁₂–tyrosine complex, several structural models (ranging from minimal to actual enzyme scaffolds) have been employed at DFT and QM/MM computations. It is shown, based on the methylmalonyl-CoA mutase (MCM) enzyme that substrate binding plays a significant role in displacing the phenoxyl proton of the tyrosine (Y89), which initiates the electron transfer from Y89 to coenzyme B₁₂. PCET-based enzymatic reaction implies that one electron-reduced form of the AdoCbl cofactor induces the cleavage of the Co–C bond, as an alternative to its neutral analogue, which can assist in understanding the origin of the observed trillion-fold rate enhancement in MCM enzyme

Co²⁺/Co⁺ Redox Tuning in Methyltransferases Induced by a Conformational Change at the Axial Ligand

Density functional theory and quantum mechanics/molecular mechanics computations predict cob­(I)­alamin (Co⁺Cbx), a universal B₁₂ intermediate state, to be a pentacoordinated square pyramidal complex, which is different from the most widely accepted viewpoint of its tetracoordinated square planar geometry. The square pyramidality of Co⁺Cbx is inspired by the fact that a Co⁺ ion, which has a dominant d⁸ electronic configuration, forms a distinctive Co⁺--H interaction because of the availability of appropriately oriented filled d orbitals. This uniquely H-bonded Co⁺Cbx may have catalytic relevance in the context of thermodynamically uphill Co²⁺/Co⁺ reduction that constitutes an essential component in a large variety of methyltransferases

Alkaloid and Polyphenol fractions of Areca nut induce TGF-β signaling in HaCaT cells.

<p>Treatment of HaCaT cells with both the Alkaloid and Polyphenol fractions of areca nut water extract induced TGF-β signaling (p-SMAD2) and its down-stream target TGM2 as shown by the western blot (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051806#pone-0051806-g003" target="_blank">Figure 3A</a>). Expression of TGF-β down-stream targets were also studied by Real Time PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051806#pone-0051806-g003" target="_blank">Figure 3B</a>) and semi quantitative PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051806#pone-0051806-g003" target="_blank">Figure 3C</a>). Induction of genes by alkaloid and polyphenol fractions of areca nut was compromised in presence of TβRI inhibitor (ALK5 inhibitor). (UN- untreated, WS- water supernatant, POL- Polyphenol supernatant, DCM- Dichloromethane fraction).</p

Electronic Structure of One-Electron-Oxidized Form of the Methylcobalamin Cofactor: Spin Density Distribution and Pseudo-Jahn–Teller Effect

The electronic and structural properties of the one-electron-oxidized form of methylcobalamin (MeCbl) cofactor have been investigated using density functional theory (DFT) and CASSCF/MC-XQDPT2 calculations. We applied two types of functionals (hybrid and GGA) which produced quite different results in terms of spin density profiles: the B3LYP description was consistent with Co­(III) and the π-cation corrin radical while the BP86 result was more in line with the Co­(IV) oxidation state. A closer inspection of both outcomes indicates that the oxidized species have a mixed π-cation corrin radical and Co­(III)/Co­(IV) character. This mixed character was further supported by high-level <i>ab initio</i> CASSCF/MC-XQDPT2 calculations, which reveal the strong mixing of the electronic states due to nondynamical correlation effects. The near degeneracy, which takes place between the ground and first excited state, was consistent with the presence of a pseudo-Jahn–Teller (pJT) effect in the oxidized form of MeCbl. In addition, the DFT-based investigation of the structurally related porphyrin complexes gives a description consistent with corrin-based analogues and reveals that the corrin species have more Co­(IV) character. The most important finding of the present study, regardless of the type of functional used, was the significant lowering of dissociation energy (∼35%), which might be due to the partial depopulation of the Co–C σ orbital upon removal of an electron

Locational comparison of essential oils from selected conifers of Himachal Pradesh

<p>Nine samples of essential oil from needles of three conifers of Pinacea family namely <i>Abies pindrow</i>, <i>Picea smithiana</i> and <i>Cedrus deodara</i> collected from three different locations of Himachal Pradesh (India) were evaluated using gas chromatography and gas chromatography-mass spectrometry. A total of 31, 17 and 13 compounds were identified from essential oil of <i>A</i>. <i>pindrow</i>, <i>P</i>. <i>smithiana</i> and <i>C</i>. <i>deodara</i>, respectively. Among the characterised components, monoterpenoid hydrocarbons were predominated. <i>α</i>-Pinene, <i>β</i>-pinene, <i>β</i>-merycene, limonene and camphene were characterised as major components. Oil of <i>C</i>. <i>deodara</i> has significant effect of location on its oil composition. Principle component analysis on gas chromatographic data reveals variation in chemical composition which may be attributed to altitude and environmental conditions.</p

Diagrammatic representation of proposed model of OSF pathogenesis by areca nut and its constituents.

<p>Areca nut first comes in contact with epithelial cells where it's both the constituent, alkaloids and polyphenols acts on the epithelial cells and induces TGF-β signaling. This induced TGF-β signaling in the epithelial cells could be source of inflammation and can also diffuse into the connective tissue where it suppresses anti-fibrogenic cytokines like BMP7. In the connective tissue, areca nut acts on fibroblast cells along with TGF-β produced from the epithelium and potentiates its action in activating fibroblast cells responsible for inducing fibrosis.</p

Activation of TGF-β Pathway by Areca Nut Constituents: A Possible Cause of Oral Submucous Fibrosis

<div><p>Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-β signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-β in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-β. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-β induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TβRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-β in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-β downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-β in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-β signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-β2 and THBS1 (activator of latent TGF-β) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-β. These data suggest a major causative role for TGF-β that is induced by areca nut in OSF progression.</p> </div